Yaping Xue

Celeste Chidiac

Yann Herault
email: herault@igbmc.fr

Claire Gaveriaux-Ruff

Pain behavior in SCN9A (Nav1.7) and SCN10A (Nav1.8) mutant rodent models

Rodent genetic models have highlighted the role of SCN9A-and SCN10A-encoded Nav1.7 and Nav1.8 voltage-gated sodium channels in pain control.

Global Scn9a knockout (KO) animals display pain insensitivity similarly to congenital insensitivity to pain in patients.

The conditional KO mouse lines allowed the assignation of Scn9a expressed in different neuronal populations in pain control. Targeting Nav1.7 and Nav1.8 constitute potential therapies for pain.

Introduction

Pain is "an unpleasant sensory and emotional experience associated with, or resembling that associated with, actual or potential tissue damage", as re-defined recently by the International Association for the Study of Pain [START_REF] Raja | The revised International Association for the Study of Pain definition of pain: concepts, challenges, and compromises[END_REF]. Pain is a leading cause of disability and disease burden [START_REF] Mills | Chronic pain: a review of its epidemiology and associated factors in population-based studies[END_REF]. As the treatment of pain is still often unsatisfactory, it is important to explore pain mechanisms for developing new therapeutic strategies [START_REF] Woolf | Capturing Novel Non-opioid Pain Targets[END_REF][START_REF] Calvo | The Genetics of Neuropathic Pain from Model Organisms to Clinical Application[END_REF].

There are four major types of pain: nociceptive pain, inflammatory pain, neuropathic pain and dysfunctional pain [START_REF] Woolf | Capturing Novel Non-opioid Pain Targets[END_REF]. njured tissues or nerves release proinflammatory molecules and nerve growth factors which lead to peripheral sensitization. The pain message is transmitted to the spinal cord via the central terminal of primary afferents and then to the pain matrix in the brain that also exerts the descending pain control [START_REF] Kuner | Cellular Circuits in the Brain and Their Modulation in Acute and Chronic Pain[END_REF].

Small fiber neuropathies (SFNs) are disorders of Aδ-fibers and C-fibers usually characterized by neuropathic pain symptoms and autonomic complaints [START_REF] Cazzato | Small fibre neuropathy[END_REF]. Interestingly, in idiopathic SFNs, nearly 30% of patients have gain-of-function (GOF) mutations in the SCN9A gene encoding for the Nav1.7 channel protein [START_REF] Faber | Gain of function Nanu1.7 mutations in idiopathic small fiber neuropathy[END_REF]. These mutations produce impaired inactivation or an enhanced resurgent current of the Nav1.7 channel and induce increased excitability in the dorsal root ganglia (DRG) neurons [START_REF] Faber | Gain of function Nanu1.7 mutations in idiopathic small fiber neuropathy[END_REF]. Nav1.7 has been investigatd as a target for analgesia but Nav1.7 inhibitors have not yet been developed as therapeutics [START_REF] Bennett | The Role of Voltage-Gated Sodium Channels in Pain Signaling[END_REF][START_REF] Alsaloum | Status of peripheral sodium channel blockers for non-addictive pain treatment[END_REF]. Additionally, SCN9A gene loss-of-function (LOF) results in congenital insensitivity to pain (CIP) [START_REF] Mcdermott | Defining the Functional Role of NaV1.7 in Human Nociception[END_REF]. Similar to SCN9A variants, several GOF mutations in the SCN10A gene encoding for the sodium channel Nav1.8 α-subunit have been reported in SFN patients, provoking neuronal hyperexcitability in the DRG neurons [START_REF] Faber | Gain-of-function Nav1.8 mutations in painful neuropathy[END_REF][START_REF] Han | The G1662S NaV1.8 mutation in small fibre neuropathy: impaired inactivation underlying DRG neuron hyperexcitability[END_REF].

Understanding the pathophysiological mechanisms underlying pain symptoms in painful SFN and other pain pathologies is important and requires preclinical models. In this review, we provide the profiles of nociceptive behaviors related to the Nav1.7 and Nav1.8 channels by evoking the different genetic models used in research until now.

The sodium voltage-gated channels Nav1.7 (SCN9A) and Nav1.8 (SCN10A)

Nav1.7 and Nav1.8 channels are voltage-gated sodium channels that play a critical role in the generation and conduction of action potentials and are important for the excitability of sensory neurons. They are composed of one α-subunit associated with one or more β-subunits. The α-subunit consists of four homologous domains (DI-DIV) consisting of six transmembrane segments [START_REF] Baker | Painful and painless mutations of SCN9A and SCN11A voltage-gated sodium channels[END_REF][START_REF] Shen | Structures of human Nav1.7 channel in complex with auxiliary subunits and animal toxins[END_REF]. The tridimensional structure and biophysical properties of these channels have been reviewed recently [START_REF] Cummins | Sodium Channels and Pain[END_REF]. Nav1.7 is a tetrodotoxin-sensitive sodium channel which opens in response to small depolarizations [START_REF] Bennett | The Role of Voltage-Gated Sodium Channels in Pain Signaling[END_REF]. Nav1.7 is preferentially expressed in the peripheral nervous system within sensory and sympathetic neurons [START_REF] Black | Expression of Nav1.7 in DRG neurons extends from peripheral terminals in the skin to central preterminal branches and terminals in the dorsal horn[END_REF][START_REF] Kanellopoulos | Mapping protein interactions of sodium channel NaV1.7 using epitope-tagged gene-targeted mice[END_REF]. The Nav1.7 protein is detected in laminae I and II of the spinal cord dorsal horn at the primary afferent terminals of sensory neurons, as shown by investigations with epitope-tagged Nav1.7 mutant mice [START_REF] Alles | Sensory neuron-derived NaV1.7 contributes to dorsal horn neuron excitability[END_REF] [START_REF] Kanellopoulos | Mapping protein interactions of sodium channel NaV1.7 using epitope-tagged gene-targeted mice[END_REF]. Nav1.8 produces a slow inactivating, tetrodotoxin-resistant current, and is known to recover rapidly from inactivation [START_REF] Bennett | The Role of Voltage-Gated Sodium Channels in Pain Signaling[END_REF][START_REF] Cummins | Sodium Channels and Pain[END_REF]. Nav1.8 contributes to the rising phase of action potential [START_REF] Cummins | Sodium Channels and Pain[END_REF]. Nav1.8 protein is expressed within the small-diameter sensory neurons of the trigeminal ganglion and DRG and in a part of medium-and large-diameter neurons as well as in lamina I and II of the spinal cord dorsal horn [8, 19-21][22].

The pain behavior analysis of genetic animal models for Scn9a and Scn10a showed that these sodium channels play important roles in acute pain sensation (nociception) and contribute to the sensitization of pain circuits, leading to hyperalgesia [START_REF] Hameed | Nav1.7 and Nav1.8: Role in the pathophysiology of pain[END_REF].

3. Role of SCN9A (Nav1.7) in pain behavior: lessons learnt from rodent models

Effect of channel absence as assessed in homozygote global KO mice and rats

Clinical genetic studies have reported that SCN9A LOF results in CIP. Genetic animal models were used to study CIP mechanisms. The impact of Nav1.7 protein absence has been assessed by the knockout (KO) Scn9a gene in mice (global KO) [START_REF] Nassar | Nociceptor-specific gene deletion reveals a major role for Nav1.7 (PN1) in acute and inflammatory pain[END_REF] [START_REF] Gingras | Global Nav1.7 knockout mice recapitulate the phenotype of human congenital indifference to pain[END_REF]. Also, partial KO rats were generated with Nav1.7 loss in DRGs, sciatic nerve, brainstem and gastrointestinal tissues but not in the olfactory tract [START_REF] Grubinska | Rat NaV1.7 loss-of-function genetic model: Deficient nociceptive and neuropathic pain behavior with retained olfactory function and intra-epidermal nerve fibers[END_REF]. These KO animals were profiled for pain behavior.

Effect on nociceptive behavior

Measurements of reflex behaviors to thermal and mechanical stimuli as well as more integrated or spontaneous pain tests were used to examine pain-like responses [START_REF] Barrot | Tests and models of nociception and pain in rodents[END_REF][START_REF] Tappe-Theodor | A common ground for pain and depression[END_REF]. Supraspinal responses to a noxious heat stimulus can be assessed by the hot plate test with paw reaction latency. Paw withdrawal latencies of wild-type (WT) and heterozygous WT/KO (HET) mice decreased with increasing temperature in the hot plate assay from 48 to 55°C while Scn9a KO mice were insensitive to heat [START_REF] Gingras | Global Nav1.7 knockout mice recapitulate the phenotype of human congenital indifference to pain[END_REF] (Table 1). Sensitivity to mechanical stimuli was evaluated using Von Frey filaments for touch, tail clip, Randall-Selitto and a pressure analgesimeter to apply pressure application to paw and tail to assess allodynia and hyperalgesia [START_REF] Barrot | Tests and models of nociception and pain in rodents[END_REF][START_REF] Tappe-Theodor | A common ground for pain and depression[END_REF].

Global Scn9a KO mice were insensitive to pressure pain in the tail clip assay. However, in the Von Frey test, the KOs showed touch sensitivity similar to that of control mice [START_REF] Gingras | Global Nav1.7 knockout mice recapitulate the phenotype of human congenital indifference to pain[END_REF]. The Scn9a LOF rats had no or faint responses to noxious chemical, thermal and prick stimuli [START_REF] Grubinska | Rat NaV1.7 loss-of-function genetic model: Deficient nociceptive and neuropathic pain behavior with retained olfactory function and intra-epidermal nerve fibers[END_REF][START_REF] Chen | Pharmacological characterization of a rat Nav1.7 loss-of-function model with insensitivity to pain[END_REF] (Table 1).

Effect on experimentally-induced pain

The formalin test is used for testing pain behaviors to formalin as a chemical irritant (phase I, 0-5 min) and subsequent acute inflammatory hyperalgesia (phase II, 10-40 min). The global Scn9a KO mice and LOF rats showed a strong reduction or no response in phases I and II in the formalin test [START_REF] Gingras | Global Nav1.7 knockout mice recapitulate the phenotype of human congenital indifference to pain[END_REF][START_REF] Grubinska | Rat NaV1.7 loss-of-function genetic model: Deficient nociceptive and neuropathic pain behavior with retained olfactory function and intra-epidermal nerve fibers[END_REF][START_REF] Chen | Pharmacological characterization of a rat Nav1.7 loss-of-function model with insensitivity to pain[END_REF]. The Complete Freund's adjuvant (CFA) model is aimed at studying chronic inflammatory hyperalgesia in rodents. WT and Scn9a KO HET control mice showed expected inflammatory hypersensitivity while whole KO mice displayed no pain-like behaviors [START_REF] Gingras | Global Nav1.7 knockout mice recapitulate the phenotype of human congenital indifference to pain[END_REF] (Table 1). The Scn9a LOF rats did not experience hypersensitivity to experimentally-induced neuropathy [START_REF] Grubinska | Rat NaV1.7 loss-of-function genetic model: Deficient nociceptive and neuropathic pain behavior with retained olfactory function and intra-epidermal nerve fibers[END_REF]. In summary, Scn9a LOF induced insensitivity or strongly reduced sensitivity in rodents, a phenotype highly analogous to CIP patients.

Effect on Scn9a deletion in specific cell populations as assessed in conditional KO mice

To study the role of Nav1.7 in specific types of neurons, several mouse lines with conditional deletion of Scn9a have been generated using the Cre-Lox system. Mice with a floxed Scn9a gene (Scn9a lox/lox ) mice were created. This review will use the simplified name Scn9a fl/fl . The Scn9a fl/fl mice were crossed with different Cre mouse lines for Scn9a gene inactivation in different cell populations. The Scn9a fl/fl mice were crossed with Scn10a cre/+ mice to obtain the Scn9a conditional KO (cKO) in primary nociceptive neurons [START_REF] Nassar | Nociceptor-specific gene deletion reveals a major role for Nav1.7 (PN1) in acute and inflammatory pain[END_REF][START_REF] Minett | Distinct Nav1.7-dependent pain sensations require different sets of sensory and sympathetic neurons[END_REF][START_REF] Hockley | Visceral and somatic pain modalities reveal NaV 1.7-independent visceral nociceptive pathways[END_REF][START_REF] Minett | Significant determinants of mouse pain behaviour[END_REF][START_REF] Shields | Sodium channel Na(v)1.7 is essential for lowering heat pain threshold after burn injury[END_REF][START_REF] Minett | Pain without nociceptors? Nav1.7-independent pain mechanisms[END_REF][START_REF] Zhou | Spider venom-derived peptide induces hyperalgesia in Nav1.7 knockout mice by activating Nav1.9 channels[END_REF] (the simplified name in this review will be Scn10a Cre -Scn9a fl/fl ). Scn9a fl/fl mice were also crossed with Avil cre/+ mice to obtain Scn9a cKO in all sensory neurons [START_REF] Minett | Distinct Nav1.7-dependent pain sensations require different sets of sensory and sympathetic neurons[END_REF][START_REF] Minett | Significant determinants of mouse pain behaviour[END_REF][START_REF] Minett | Pain without nociceptors? Nav1.7-independent pain mechanisms[END_REF][START_REF] Minett | Endogenous opioids contribute to insensitivity to pain in humans and mice lacking sodium channel Nav1.7[END_REF]. The name of this line in this review will be Avil Cre -Scn9a fl/fl . In addition, Scn9a fl/fl mice were crossed with Wnt1 cre/+ mice for Scn9a conditional gene inactivation in sensory and sympathetic neurons [START_REF] Minett | Distinct Nav1.7-dependent pain sensations require different sets of sensory and sympathetic neurons[END_REF][START_REF] Minett | Significant determinants of mouse pain behaviour[END_REF][START_REF] Minett | Pain without nociceptors? Nav1.7-independent pain mechanisms[END_REF][START_REF] Pereira | Analgesia linked to Nav1.7 loss of function requires micro-and deltaopioid receptors[END_REF]. In this review these cKO mice will be referred to as Wnt1 Cre -Scn9a fl/fl . Moreover, Scn9a gene inactivation could be induced at the adult age. Scn9a fl/fl mice were crossed with mice where the Cre-ERT transgene was driven by the CMV early enhancer/chicken beta-actin promoter, allowing Cre recombinase activation upon tamoxifen induction [START_REF] Shields | Insensitivity to Pain upon Adult-Onset Deletion of Nav1.7 or Its Blockade with Selective Inhibitors[END_REF]. This cKO line will be named pCAGG CreERT -Scn9a fl/fl . Following tamoxifen induction, Scn9a expression was abolished in the DRG (mRNA, protein), trigeminal ganglia (mRNA) and sympathetic superior cervical ganglia (mRNA) in pCAGG CreERT -Scn9a fl/fl mice [START_REF] Shields | Insensitivity to Pain upon Adult-Onset Deletion of Nav1.7 or Its Blockade with Selective Inhibitors[END_REF].

Effect on nociceptive behavior

Sensitivity to heat noxious stimuli was determined in the Hargreaves and hot plate tests. Altogether, the Scn9a cKO lines were less sensitive in the Hargreaves test. The Scn10a Cre -Scn9a fl/fl line showed decreased sensitivity with the slow ramp [START_REF] Nassar | Nociceptor-specific gene deletion reveals a major role for Nav1.7 (PN1) in acute and inflammatory pain[END_REF][START_REF] Minett | Distinct Nav1.7-dependent pain sensations require different sets of sensory and sympathetic neurons[END_REF][START_REF] Minett | Significant determinants of mouse pain behaviour[END_REF][START_REF] Zhou | Spider venom-derived peptide induces hyperalgesia in Nav1.7 knockout mice by activating Nav1.9 channels[END_REF] while the Avil Cre -Scn9a fl/fl , Wnt1 Cre -Scn9a fl/fl and pCAGG CreERT -Scn9a fl/fl lines showed behavioral response deficits to both fast and slow heat ramps [START_REF] Minett | Distinct Nav1.7-dependent pain sensations require different sets of sensory and sympathetic neurons[END_REF][START_REF] Minett | Significant determinants of mouse pain behaviour[END_REF][START_REF] Pereira | Analgesia linked to Nav1.7 loss of function requires micro-and deltaopioid receptors[END_REF][START_REF] Shields | Insensitivity to Pain upon Adult-Onset Deletion of Nav1.7 or Its Blockade with Selective Inhibitors[END_REF], suggesting a differential contribution of Nav1.7 in the different neuron types for mediating the response to transduced heat [START_REF] Minett | Significant determinants of mouse pain behaviour[END_REF]. Furthermore, in the more integrated hot plate test test, only the Wnt1 Cre -Scn9a fl/fl and pCAGG CreERT -Scn9a fl/fl mice showed a pronounced response attenuation [START_REF] Minett | Distinct Nav1.7-dependent pain sensations require different sets of sensory and sympathetic neurons[END_REF][START_REF] Pereira | Analgesia linked to Nav1.7 loss of function requires micro-and deltaopioid receptors[END_REF][START_REF] Shields | Insensitivity to Pain upon Adult-Onset Deletion of Nav1.7 or Its Blockade with Selective Inhibitors[END_REF] (Table 1).

Response to cooling and extreme cold were measured by the acetone and cold plate tests [START_REF] Barrot | Tests and models of nociception and pain in rodents[END_REF]. The cKO lines displayed normal response on the cold plate in contrast to LOF rats that were less sensitive. In the acetone test, all the cKO lines showed deficits in cooling perception except the Scn10a Cre -Scn9a fl/fl line [START_REF] Minett | Distinct Nav1.7-dependent pain sensations require different sets of sensory and sympathetic neurons[END_REF] (Table 1). This was corroborated by the attenuated response of Avil Cre -Scn9a fl/fl mice to cooling in the dynamic thermal place preference test [START_REF] Minett | Significant determinants of mouse pain behaviour[END_REF] (Table 1). Altogether, these results on Scn9a cKO lines suggest that Nav1.7 expressed on Nav1.8 negative cells are implicated in cooling (acetone) response.

The cKO lines displayed pronounced analgesia to noxious pressure in the Randall-Selitto test (Table 1). The Scn10a Cre -Scn9a fl/fl mice showed normal response to touch in six articles from three laboratories [START_REF] Nassar | Nociceptor-specific gene deletion reveals a major role for Nav1.7 (PN1) in acute and inflammatory pain[END_REF][START_REF] Minett | Distinct Nav1.7-dependent pain sensations require different sets of sensory and sympathetic neurons[END_REF][START_REF] Minett | Significant determinants of mouse pain behaviour[END_REF][START_REF] Minett | Pain without nociceptors? Nav1.7-independent pain mechanisms[END_REF][START_REF] Shields | Insensitivity to Pain upon Adult-Onset Deletion of Nav1.7 or Its Blockade with Selective Inhibitors[END_REF][START_REF] Liu | NGF-Induced Nav1.7 Upregulation Contributes to Chronic Post-surgical Pain by Activating SGK1-Dependent Nedd4-2 Phosphorylation[END_REF] except when they were tested with the Electronic von Frey Anesthesiometer [START_REF] Zhou | Spider venom-derived peptide induces hyperalgesia in Nav1.7 knockout mice by activating Nav1.9 channels[END_REF]. This difference may have been due to the continuous force application and represent a pressure rather than a touch stimulus, thereby consolidating the data obtained with the Randall-Selitto test. Interestingly, both Avil Cre -Scn9a fl/fl and Wnt1 Cre -Scn9a fl/fl mice but not Scn10a Cre -Scn9a fl/fl mice showed deficits in response to von Frey filaments applied to hairy skin [START_REF] Minett | Significant determinants of mouse pain behaviour[END_REF] (Table 1), revealing mechanosensation by hair follicles dependent on Nav1.7 mainly in Nav1.8-negative neurons [START_REF] Minett | Significant determinants of mouse pain behaviour[END_REF].

Effect on experimentally-induced pain

The role of Nav1.7 on Nav1.8-positive neurons in visceral pain was investigated with the Scn10a Cre -Scn9a fl/fl mice [START_REF] Hockley | Visceral and somatic pain modalities reveal NaV 1.7-independent visceral nociceptive pathways[END_REF]. Scn10a Cre -Scn9a fl/fl animals had normal sensitivity, while pCAGG CreERT -Scn9a fl/fl mice displayed a decreased response in the acetic-acid-induced model [START_REF] Shields | Insensitivity to Pain upon Adult-Onset Deletion of Nav1.7 or Its Blockade with Selective Inhibitors[END_REF]. Together, this suggests that that Nav1.7 in neurons other than Nav1.8 neurons contributed to visceral pain, while Nav1.8 was shown to contribute to visceral pain (see section 4.2.).

The Scn9a cKO lines were assessed for inflammatory pain. Both Scn10a Cre -Scn9a fl/fl and Avil Cre -Scn9a fl/fl lines displayed reduced response in the two formalin test phases [START_REF] Nassar | Nociceptor-specific gene deletion reveals a major role for Nav1.7 (PN1) in acute and inflammatory pain[END_REF][START_REF] Minett | Distinct Nav1.7-dependent pain sensations require different sets of sensory and sympathetic neurons[END_REF], while pCAGG CreERT -Scn9a fl/fl mice had an attenuated response in phase I only [START_REF] Shields | Insensitivity to Pain upon Adult-Onset Deletion of Nav1.7 or Its Blockade with Selective Inhibitors[END_REF]. In the carrageenan inflammatory model, thermal hyperalgesia was absent in Scn10a Cre -Scn9a fl/fl mice [START_REF] Nassar | Nociceptor-specific gene deletion reveals a major role for Nav1.7 (PN1) in acute and inflammatory pain[END_REF] (Table 1). In the CFA model, heat hypersensitivity was abolished in both Scn10a Cre -Scn9a fl/fl and pCAGG CreERT -Scn9a fl/fl animals while mechanical allodynia was reduced only in the latter mice [START_REF] Shields | Insensitivity to Pain upon Adult-Onset Deletion of Nav1.7 or Its Blockade with Selective Inhibitors[END_REF]. Altogether, these results highlighted the contribution of Nav1.7 in peripheral neurons in inflammatory hyperalgesia.

Neuropathic pain is caused by neuronal injury in the peripheral or central nervous system, and preclinical models have been developed [START_REF] Barrot | Tests and models of nociception and pain in rodents[END_REF]. Chronic constriction injury (CCI), spinal nerve transection (SNT), partial sciatic nerve ligation (pSNL) and spared-nerve injury (SNI) were utilized as nerve injury models in the Scn9a cKO mouse lines [START_REF] Minett | Distinct Nav1.7-dependent pain sensations require different sets of sensory and sympathetic neurons[END_REF][START_REF] Minett | Significant determinants of mouse pain behaviour[END_REF][START_REF] Shields | Insensitivity to Pain upon Adult-Onset Deletion of Nav1.7 or Its Blockade with Selective Inhibitors[END_REF][START_REF] Nassar | Neuropathic pain develops normally in mice lacking both Na(v)1.7 and Na(v)1.8[END_REF]. The Scn10a Cre -Scn9a fl/fl mice developed mechanical but attenuated cold allodynia following CCI or pSNL, while they showed normal allodynia after SNT (Table 1). The Avil Cre -Scn9a fl/fl mice showed cold and mechanical allodynia during SNT but attenuated allodynia following CCI (Table 1).

Deleting Scn9a in both sensory and sympathetic neurons (Wnt1 Cre -Scn9a fl/fl ) abolished allodynia upon SNT or CCI (Table 1). In pCAGG CreERT -Scn9a fl/fl mice, SNI induced weak cold allodynia but normal mechanical hypersensitivity [START_REF] Shields | Insensitivity to Pain upon Adult-Onset Deletion of Nav1.7 or Its Blockade with Selective Inhibitors[END_REF]. In Avil Cre -Scn9a fl/fl and Wnt1 Cre -Scn9a fl/fl mice, oxaliplatin-induced neuropathic pain and cancer-induced pain developed as in control littermates (Table 1). Furthermore, in a burn injury model, Nav1.7 in Nav1.8-positive neurons contributed to heat but not mechanical hyperalgesia [START_REF] Shields | Sodium channel Na(v)1.7 is essential for lowering heat pain threshold after burn injury[END_REF] (Table 1).

Altogether, the behavioral analyses of KO and cKO animals showed that Nav1.7 is a major contributor to both acute nociception and experimentally-induced chronic hyperalgesia and revealed the role of Nav1.7 in specific neuronal populations in the different pain models.

Nav1.7 and the opioid pathway

LOF mutations in humans or global Scn9a KO in mice or rats lead to insensitivity to pain. The Preproenkephalin (Penk) gene encodes for the endogenous opioid peptide enkephalin, an endogenous paincontroller. Interestingly, Penk mRNA was found to be elevated in Avil Cre -Scn9a fl/fl DRGs. The opioid antagonist naloxone reversed hyposensitivity in a SCN9A-null CIP patient and in Avil Cre -Scn9a fl/fl mice [START_REF] Minett | Endogenous opioids contribute to insensitivity to pain in humans and mice lacking sodium channel Nav1.7[END_REF]. Penk expression was also increased in Wnt1 Cre -Scn9a fl/fl animals. Wnt1 Cre -Scn9a fl/fl mice were crossed mu (MOR) or delta (DOR) opioid receptor KO mice, and Scn9a KO-driven analgesia was abolished in triple Wnt1 Cre -Scn9a fl/fl /MOR/DOR KO mice [START_REF] Pereira | Analgesia linked to Nav1.7 loss of function requires micro-and deltaopioid receptors[END_REF]. Altogether, this suggested that endogenous opioids contribute to analgesia driven by Scn9a gene inactivation. However, naloxone did not reverse analgesia in Scn9a LOF rats [START_REF] Chen | Pharmacological characterization of a rat Nav1.7 loss-of-function model with insensitivity to pain[END_REF] and PENK was not expressed or up-regulated in sensory neurons differentiated from a patient with CIP [START_REF] Mcdermott | Defining the Functional Role of NaV1.7 in Human Nociception[END_REF].

Hence, the link between loss of Nav1.7 and the opioid system in CIP is still a matter of debate. The mouse mutant termed Possum which carries a T790A hypermorphic mutation of the Scn10a gene was identified following N-ethyl-N-nitrosourea mutagenesis. These mutants have posture abnormalities and deficits [START_REF] Blasius | Hypermorphic mutation of the voltage-gated sodium channel encoding gene Scn10a causes a dramatic stimulus-dependent neurobehavioral phenotype[END_REF]. They had normal responses to Von Frey filaments but an increased response to a needle prick on the hindpaw [41, 42][42]. They displayed increased sensitivity on the cold plate at -1°C but normal sensitivity on the hot plate [START_REF] Blasius | Hypermorphic mutation of the voltage-gated sodium channel encoding gene Scn10a causes a dramatic stimulus-dependent neurobehavioral phenotype[END_REF]. Following mustard oil application to the hindpaw, Possum mice remained motionless [START_REF] Garrison | A gain-of-function voltage-gated sodium channel 1.8 mutation drives intense hyperexcitability of A-and C-fiber neurons[END_REF], the relationship of this behavior to pain remaining unknown. In the CFA inflammatory model, Possum mice showed normal mechanical allodynia [START_REF] Blasius | Hypermorphic mutation of the voltage-gated sodium channel encoding gene Scn10a causes a dramatic stimulus-dependent neurobehavioral phenotype[END_REF] (Table 2). In summary, the Possum mice had hyperactive Nav1.8 channels and showed increased sensitivity to extreme noxious mechanical and cold stimuli but not to the other pain challenges.

Role of

Effect of Nav1.8 channel absence as assessed in global KO mice

Effect on nociceptive behavior and spontaneous pain

The studies on Scn10a KO mice revealed deficits in both mechanical and thermal perception (Table 2). Scn10a KO mice showed analgesia to noxious mechanical pressure [START_REF] Minett | Significant determinants of mouse pain behaviour[END_REF][START_REF] Nassar | Neuropathic pain develops normally in mice lacking both Na(v)1.7 and Na(v)1.8[END_REF][START_REF] Akopian | The tetrodotoxin-resistant sodium channel SNS has a specialized function in pain pathways[END_REF], but no change in reaction to touch in the Von Frey test [START_REF] Shields | Sodium channel Na(v)1.7 is essential for lowering heat pain threshold after burn injury[END_REF][START_REF] Minett | Pain without nociceptors? Nav1.7-independent pain mechanisms[END_REF][START_REF] Nassar | Neuropathic pain develops normally in mice lacking both Na(v)1.7 and Na(v)1.8[END_REF][START_REF] Akopian | The tetrodotoxin-resistant sodium channel SNS has a specialized function in pain pathways[END_REF][START_REF] Kerr | A role for the TTX-resistant sodium channel Nav 1.8 in NGF-induced hyperalgesia, but not neuropathic pain[END_REF][START_REF] Leo | Exploring the role of nociceptor-specific sodium channels in pain transmission using Nav1.8 and Nav1.9 knockout mice[END_REF]. Concerning responses to thermal stimuli, Scn10a KO animals showed increased withdrawal latency to slow but not to fast heat ramps in the Hargreaves test [START_REF] Minett | Significant determinants of mouse pain behaviour[END_REF]. The same analgesia was found in Scn10a KO mice when using the slow heating condition in the paw flick and tail flick tests [START_REF] Akopian | The tetrodotoxin-resistant sodium channel SNS has a specialized function in pain pathways[END_REF] but not in other studies with fast heating [START_REF] Nassar | Neuropathic pain develops normally in mice lacking both Na(v)1.7 and Na(v)1.8[END_REF][START_REF] Kerr | A role for the TTX-resistant sodium channel Nav 1.8 in NGF-induced hyperalgesia, but not neuropathic pain[END_REF][START_REF] Leo | Exploring the role of nociceptor-specific sodium channels in pain transmission using Nav1.8 and Nav1.9 knockout mice[END_REF]. Accordingly, a slow temperature ramp allowed detecting analgesia in Scn10a KO mice. Scn10a KO mice were less sensitive in the tail-flick test [START_REF] Akopian | The tetrodotoxin-resistant sodium channel SNS has a specialized function in pain pathways[END_REF] but normally sensitive in the hot plate test integrated in all the temperatures tested [START_REF] Nassar | Neuropathic pain develops normally in mice lacking both Na(v)1.7 and Na(v)1.8[END_REF][START_REF] Akopian | The tetrodotoxin-resistant sodium channel SNS has a specialized function in pain pathways[END_REF][START_REF] Leo | Exploring the role of nociceptor-specific sodium channels in pain transmission using Nav1.8 and Nav1.9 knockout mice[END_REF]. Scn10a deficient mice also revealed a reduced response to extreme but not to mild cold [START_REF] Luiz | Cold sensing by NaV1.8-positive and NaV1.8-negative sensory neurons[END_REF][START_REF] Zimmermann | Sensory neuron sodium channel Nav1.8 is essential for pain at low temperatures[END_REF] (Table 2) and were less sensitive in visceral pain models [START_REF] Laird | Deficits in visceral pain and referred hyperalgesia in Nav1.8 (SNS/PN3)null mice[END_REF].

Effect on experimentally-induced pain

The Scn10a KO animals were tested in both inflammatory and neuropathic models. The response of mutant mice to formalin was similar to that of control mice. Following carrageenan inflammation, hyperalgesia onset was slightly delayed in Scn10a KO mice [START_REF] Akopian | The tetrodotoxin-resistant sodium channel SNS has a specialized function in pain pathways[END_REF]. In the CFA model, mutant mice displayed both thermal and mechanical hyperalgesia, with faster recovery from heat pain [START_REF] Leo | Exploring the role of nociceptor-specific sodium channels in pain transmission using Nav1.8 and Nav1.9 knockout mice[END_REF]. These results suggest a role of Nav1.8 in the kinetics of inflammatory pain but not in lowering nociceptive thresholds. The null mutants developed heat and mechanical hypersensitivity following burn injury [START_REF] Shields | Sodium channel Na(v)1.7 is essential for lowering heat pain threshold after burn injury[END_REF] and SNT [START_REF] Minett | Significant determinants of mouse pain behaviour[END_REF]. Scn10a KO mice showed attenuated cold allodynia in the SNI model [START_REF] Leo | Exploring the role of nociceptor-specific sodium channels in pain transmission using Nav1.8 and Nav1.9 knockout mice[END_REF] and reduced cold allodynia to CCI injury in one out of two reports [START_REF] Minett | Significant determinants of mouse pain behaviour[END_REF][START_REF] Leo | Exploring the role of nociceptor-specific sodium channels in pain transmission using Nav1.8 and Nav1.9 knockout mice[END_REF] (Table 2). Taken globally, the Nav1.8 channel contributes to cold allodynia in neuropathic pain models.

Effect on the channel as assessed in Nav1.8-Cre mice

Knock-in mouse lines that express Cre-recombinase driven by the Scn10a locus were generated [START_REF] Agarwal | Conditional gene deletion in primary nociceptive neurons of trigeminal ganglia and dorsal root ganglia[END_REF][START_REF] Stirling | Nociceptor-specific gene deletion using heterozygous NaV1.8-Cre recombinase mice[END_REF][START_REF] Nassar | Nociceptor-specific gene deletion reveals a major role for Nav1.7 (PN1) in acute and inflammatory pain[END_REF] to develop conditional KO mice with nociceptive neurons, Scn10a Cre/+ . These mice had normal behavior in the mechanical and heat pain tests [START_REF] Agarwal | Conditional gene deletion in primary nociceptive neurons of trigeminal ganglia and dorsal root ganglia[END_REF][START_REF] Stirling | Nociceptor-specific gene deletion using heterozygous NaV1.8-Cre recombinase mice[END_REF][START_REF] Nassar | Nociceptor-specific gene deletion reveals a major role for Nav1.7 (PN1) in acute and inflammatory pain[END_REF](Table 2). Inflammatory and SNL-induced neuropathic pain developed normally in Scn10a Cre/+ mice [START_REF] Nassar | Nociceptor-specific gene deletion reveals a major role for Nav1.7 (PN1) in acute and inflammatory pain[END_REF]. [START_REF] Stirling | Nociceptor-specific gene deletion using heterozygous NaV1.8-Cre recombinase mice[END_REF] (Table 2). Therefore, as the Scn10a Cre/+ mice behaved as WT mice in the pain models, they were used to conditionally inactivate their genes of interest in nociceptive neurons.

Perspectives

The genetic rodent models described here, together with pharmacological approaches that are beyond the scope of this short review, showed that Nav1.7 and Nav1.8 play important roles in nociception and chronic hyperalgesia. Two teams have developed sensory neurons differentiated from patient-derived Induced Pluripotent Stem Cells that model inherited erythromelalgia [START_REF] Cao | Pharmacological reversal of a pain phenotype in iPSC-derived sensory neurons and patients with inherited erythromelalgia[END_REF][START_REF] Mis | Resilience to Pain: A Peripheral Component Identified Using Induced Pluripotent Stem Cells and Dynamic Clamp[END_REF][START_REF] Meents | The role of Nav1.7 in human nociceptors: insights from human induced pluripotent stem cell-derived sensory neurons of erythromelalgia patients[END_REF]. This novel preclinical model provides a platform which enables the assessment of the sodium channel function in vitro in cells from patients. GOF mutants of SCN9A and SCN10A have been reported in SFN patients with chronic pain while LOF mutants of SCN9A lead to a loss of pain perception in CIP patients. These data suggest that SCN9A is mandatory for pain in humans, and that elucidating the role of SCN10A requires more investigation. Novel rodent genetic models for the GOF mutations found in SFN patients could be generated by homologous recombination or CRISPR-Cas9 techniques. Also, the other sodium channels including the Nav1.9 channel playing important roles in pain and mutations in these channels have been identified [START_REF] Calvo | The Genetics of Neuropathic Pain from Model Organisms to Clinical Application[END_REF][START_REF] Bennett | The Role of Voltage-Gated Sodium Channels in Pain Signaling[END_REF][START_REF] Baker | Painful and painless mutations of SCN9A and SCN11A voltage-gated sodium channels[END_REF][START_REF] Cummins | Sodium Channels and Pain[END_REF]. Genetic models for mutations in these other genes will allow improving understanding of their roles in chronic pain.

The evaluation of reflexive pain behavior has been applied in preclinical pain research for decades. Present and future genetic models could be tested in more clinically relevant approaches of spontaneous pain behavior, the attenuation of pathological pain, and restoring functions that are decreased in pain patients [START_REF] Barrot | Tests and models of nociception and pain in rodents[END_REF][START_REF] Tappe-Theodor | A common ground for pain and depression[END_REF]. In addition, exploring the roles of Scn genes in pain behavior would necessitate the study of mutations in different genetic backgrounds and in both sexes [START_REF] Mogil | Laboratory environmental factors and pain behavior: the relevance of unknown unknowns to reproducibility and translation[END_REF]. More globally, the combination of genetic models and other approaches including genetic screening will allow identifying additional components of chronic pain [START_REF] Calvo | The Genetics of Neuropathic Pain from Model Organisms to Clinical Application[END_REF].

Summary

This review summarized the literature published on pain behavior in Scn9a and Scn10a genetic rodent models. Deleting Scn9a and Scn10a either in the whole body or in specific neuron populations attenuates pain responses, mirroring the clinical features of CIP patients. The analysis of genetic animal models for the mutations found in SFN patients would reveal how these mutations mediate SFN-related pathology including pain.

Mechanical

4 . 1 .

 41 Nav1.8 in pain behavior: lessons learnt from mouse models Pain behavior in the Scn10a T790A possum mice.

Table 1 .

 1 Pain Behaviors in Scn9a(Nav1.7)-related genetic models Chronic Constriction Injury; CFA: Complete Freund's Adjuvant; KO, knockout; LOF: Loss-of-function; na: not available; SNI: Spared Nerve Injury; pSNL: partial sciatic Nerve Ligation; SNL: Spinal Nerve Ligation; SNT: Spinal Nerve Transection; =: no change; : less sensitive compared to control

		Scn9a LOF rats;	Scn10a Cre -Scn9a fl/fl ;	Wnt1 Cre -Scn9a fl/fl ;
		Scn9a Global KO	Advill Cre -Scn9a fl/fl	pCAGG CreERT -Scn9a fl/fl
		[25, 26, 29]	[24, 30-35, 39]	[30, 32, 34, 37-38]
		Basal nociception
	Mechanical
	Von Frey	=; =	=; =	= ; =
	Randall-Selitto or tail clip	na;
	Pin-prink	; na	na	na
	Heat
	Hargreaves	; na	=;
	Hot Plate or tail immersion		=
	Cold
	Acetone		=;	; na
	Cold Plate or Dry ice		=	=; na
	Dynamic thermal place preference, cooling	=	na;	na;
		Inflammatory pain
	Formalin Phase I		=	ND;
	Formalin Phase II			ND; =
	CFA or Carrageenan
	Hargreaves	na;	; na	na;
	Von Frey	na	; na	na; =
		Neuropathic pain
	SNT
	Acetone	na	=	; na
	Von Frey	na	=	; na
	CCI
	Acetone	na		; na
	Von Frey	na	= ;	; na
	pSNL, SNL or SNI
	Acetone	na	; na	na;
	Von Frey	; na	= ; na	na; =
	Oxaliplatin
	Acetone	na	=	=; na
	Von Frey	na	=	=; na
		Other pain models
	Cancer-induced pain	na	na; =	=; na
	Burn injury
	Hargreaves	na	; na	na
	Von Frey	na	=; na	na

Table 2 .

 2 Pain Behaviors in Scn10a (Nav1.8)-related genetic models CCI: Chronic Constriction Injury; CFA: Complete Freund's Adjuvant; KO: knockout; na: not available; SNI: Spared Nerve Injury; SNL: Spinal Nerve Ligation; SNT: Spinal Nerve Transection =: no change; less sensitive compared to control.

	Scn10a Global KO	Scn10a Cre/+
	[32-34, 40, 43, 44-48]	[19-20, 24]
	Basal nociception

Acknowledgment

We would like to thank members of the research group, of the IGBMC laboratory and of the ICS. This project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 721841-PAIN-NET. This work has been also supported by the National Centre for Scientific Research (CNRS), the French National Institute of Health and Medical Research (INSERM), the University of Strasbourg (Unistra), the French state funds through the "Agence Nationale de la Recherche" under the frame programme Investissements d'Avenir [ANR-10-IDEX-0002-02,

* equal contribution $ equal last authors

Support and grant information

This work has been funded by the European commission H2020 programme (grant number -721841 -PAIN-Net).


Eduardo Robles

René Herrera

Pedro Luis De Hoyos Martínez

Javier Fernández Rodríguez

Jalel Labidi

Pedro Luis

Pedro L De Hoyos Martínez

Valorization of heat-treated wood after service life through a cascading process for the production of lignocellulosic derivatives

Keywords: heat-treatment, organosolv lignin, cellulose, wood products, fractionation, cascade approach

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The use of wood-based products in Europe is projected to increase threefold worldwide between 2010 and 2050 [START_REF] Dammer | Cascading Use of Wood Products[END_REF]. In this frame, the inventories of Monterey pine (Pinus radiata D. Don) in the Basque Country, where is located this research work, has reported an increase in wood production from 15 million m 3 in 1976 to more than 27 million m 3 in 2017 (Etxabe Villasante, 2018). This fact has awakened considerable interest in using this vital resource in high value-added applications after its modification, such as building applications after its thermal or chemical treatment, elaboration of biomaterials, plywood, fiberboards, among others [START_REF] Wei | Structure, mechanical performance, and dimensional stability of radiata pine (Pinus radiata D. Don) scrimbers[END_REF]. Monterey pine is the species that occupy the most significant area (31 % of the total wooden forest area) and about 85-90 % greater forest productivity in the Basque Country. Basque forests can be considered one of the lands with the highest concentrations of Monterey pine in the northern hemisphere; it should be regarded as an essential asset for the local economy [START_REF] Cantero | El futuro del pino radiata en el País Vasco[END_REF]. However, the highly fractioned size of private-owned forestlands, the aging of the local population and the strong regulations to cut down have supposed a challenge to the exploitation of pine by the local owners, which often cut down the trees, then leave them as green timber. In this sense, a proficient value-added application has been installing a kiln by local entrepreneurs, who have developed a thermal modification process marketed as Thermogenik®. This method has treated local wood samples for outdoor applications such as roofing, decking, and façades. The performance of thermally modified woods has been deeply studied for several applications, showing outstanding results [START_REF] Herrera | Evolution of thermally modified wood properties exposed to natural and artificial weathering and its potential as an element for façades systems[END_REF]. However, in the frame of demands and projections of the XXI century, the mere upgrading of wood or timber as a material is not enough. The thermal modification of wood is an industrial process that take place at temperatures between 170-220 °C in a reduced oxygen atmosphere (e.g. steam saturated, gas, oil) in order to prevent oxidative combustion of the wood components. The thermally modified wood (TMW) presents better dimensional stability, hydrophobicity, and protection against rot fungi [START_REF] Jones | View of A Review of Wood Modification Globally -Updated Findings from COST FP1407 | Interdisciplinary Perspectives on the Built Environment[END_REF]. The most notable characteristic of the TMW is its color change to darker tones, and durability, which can considerably improve the wood durability class depending on the type of wood and the treatment conditions. The changes in the wood during thermal modification were studied in last decades and several patents and parent were developed. According to the European Committee for Standardization (European Committee for Standardization, 2008) TMW is wood in which the composition of the cell-wall material and its physical properties have been modified by exposure to a temperature higher than 160 °C with limited access to oxygen. The main changes in the wood matrix are the redistribution of lignin components, the degradation of hemicelluloses caused by deacetylation and the acid catalyzed hydrolysis of the polysaccharide chains, cross-linking and repolymerization reactions with different characteristics depending on the species, atmosphere, and temperature [START_REF] Herrera-Díaz | Influence of the wood quality and treatment temperature on the physical and mechanical properties of thermally modified radiata pine[END_REF][START_REF] Jones | View of A Review of Wood Modification Globally -Updated Findings from COST FP1407 | Interdisciplinary Perspectives on the Built Environment[END_REF][START_REF] Li | Response of hygroscopicity to heat treatment and its relation to durability of thermally modified wood[END_REF]. The assessment of the process-related environmental impact of heat treatments has been limited; this can be because they lack chemicals and have encouraged the assumption that heat treatments are intrinsically more eco-friendly. According to Sandberg and Kutnar, TMW materials can mitigate climate change and promote sustainable development by reducing energy intake, solid and volatile emissions, reducing pollution, and ecosystem damage [START_REF] Sandberg | Thermal Modified Timber (Tmt): Recent Development in Europe and North America[END_REF]. Despite the industrial expansion of heat treatments, environmental guidelines are becoming more restrictive, and there is an increase in the sensitivity and awareness of the market to the environmental performance of products and services, preferring those that are eco-friendly. At the present stage, it is necessary to consider the environmental performance of the thermally modified products and end-of-life scenarios to create strategies after the end of life of products. In general, to develop an environmental performance of a product is necessary to include recycling, upcycling, the cradle-to-cradle/cradleto-gate paradigm, and end-of-life disposal options. Several studies on ecological solutions in the process stage are performed, mainly focused on releasing volatile compounds into the atmosphere, including recovery by condensation, purification, and successive filtration processes (Herrera et al., 2016a). Additionally, the post-service-life reuse or disposal of any material requires strict handling, even for a bio-based material as wood. For this reason, several European countries have developed facilities for the energetic valorization of bio-based materials after their usage [START_REF] Bentsen | Biomass for energy in the European Union -A review of bioenergy resource assessments[END_REF]. Nevertheless, the life cycle assessment of wood as a material is truncated if energetic valorization is the following step after service life [START_REF] Campbell-Johnston | The circular economy and cascading: towards a framework[END_REF]. Instead, the fractionation of the lignocellulosic macro components that constitute wood: holocellulose and lignin carbohydrate and aromatic fractions could open a frame to increase the value and service of wood after its first use, following the principles requested by the circular economy concept, which are considerably demanding nowadays [START_REF] Islam | Sustainability metrics of pretreatment processes in a waste derived lignocellulosic biomass biorefinery[END_REF]. In this sense, the cascading approach is considered the most suitable method for the whole valorization of the different biomass fractions. There are three definitions of cascading processes: cascading-in-time, cascading-in-value, and cascading-in-function. Cascading in function has been described as a process through which different products can be produced simultaneously, maximizing the value of the original biomass, resembling the functionality of a biorefinery [START_REF] Keegan | Cascading use: a systematic approach to biomass beyond the energy sector[END_REF][START_REF] Olsson | Cascading of woody biomass: definitions, policies and effects on international trade[END_REF][START_REF] Sokka | Cascading use of wood in Finland-with comparison to selected EU countries[END_REF]. The most common process to fractionate the wood-based materials is the one applied for the pulp and paper industry, namely, the Kraft process, followed by an elemental chlorine-free (ECF) sequence to bleach the intermediate pulp. However, more environmentally friendly processes can be designed, such as sulfur-free pulping processes or total chlorine-free bleaching sequences (TCF) [START_REF] Oliaei | Microfibrillated lignocellulose (MFLC) and nanopaper films from unbleached kraft softwood pulp[END_REF][START_REF] Robles | Production of cellulose nanoparticles from blue agave waste treated with environmentally friendly processes[END_REF]. Concerning the pulping stage, the organosolv delignification process includes a diverse range of organic solvents to fractionate lignocellulosic biomass into rich cellulose pulp, a water-soluble hemicellulose stream, and a concentrated lignin fraction. The organic solvents triggered the solubilization of lignin without altering its chemical structure to a high degree, which has positioned these processes into the most attractive ones to preserve the native structure of lignin during the extraction without undergoing strong recondensation reactions [START_REF] Pandey | Lignin Depolymerization and Conversion: A Review of Thermochemical Methods[END_REF]. The high purity of the obtained lignin by these methods, together with the lack of sulfur in its molecular structure, allows lignin to be employed as a source of aromatic structures to produce high added-value chemicals, which can be used for the production of adhesives, binders, in polymer substitutions or as a phenolic precursor [START_REF] Zakzeski | The Catalytic Valorization of Lignin for the Production of Renewable Chemicals[END_REF][START_REF] Zhao | Organosolv fractionating pre-treatment of lignocellulosic biomass for efficient enzymatic saccharification: chemistry, kinetics, and substrate structures[END_REF]. Besides, the low boiling point of the organic solvents allows the recovery of the solvent to avoid its disposal after the process [START_REF] Fernández-Rodríguez | Direct lignin depolymerization process from sulfur-free black liquors[END_REF]. Thanks to all these advantages, it is expected that the organosolv methods may awaken more industrial interest in the future for biomass fractionation [START_REF] Da Silva | Hydrothermal and organic-chemical treatments of eucalyptus biomass for industrial purposes[END_REF]. For the bleaching stages, the TCF sequences constitute environmentally friendly alternatives to remove the phenolic chromophores from the remaining lignin fraction in the cellulosic pulp without chlorine compounds due to their adverse environmental effects [START_REF] Ibarra | Integrating laccase-mediator treatment into an industrial-type sequence for totally chlorine-free bleaching of eucalypt kraft pulp[END_REF][START_REF] Nelson | Elemental chlorine free (ECF) and totally chlorine free (TCF) bleaching of pulps[END_REF]. When doing a TCF bleaching, removing the metals becomes more critical because hydrogen peroxide is applied early in the sequence at higher charges. The elimination of the metals is improved with the addition of a chelating agent. Ethylenediaminetetracetic acid (EDTA) and diethyleneaminepentacetic acid (DTPA) compounds are the best chelating agents for double-charged metal ions. However, using either DTPA or EDTA in "green pulping and bleaching" is currently discouraged as none of these agents is biodegradable, nor are they efficiently handled, as standard water treatment plants cannot remove these components from water. Therefore, they represent considerable harm to the ecosystems in which those streams are deployed. For this reason, it is recommended to use another metal chelating agent, such as the 2,6-Pyridinedicarboxylic acid (PDA), which has been proved to be an effective chelating agent with similar results to those of EDTA [START_REF] Pinto | Pre-treatment of the paper pulp in the bleaching process using biodegradable chelating agents[END_REF]. In the present work, the cascading of local wood residues after its usage to be transformed into bio-based materials by fractionating the whole structure of the macro-components is approached. The goal is to guarantee the integral valorization of these residues by obtaining building block components capable of being transformed into high-value-added compounds. An environmentally friendly and robust process has been designed using an organic acid pretreatment, a sulfur-free delignification stage, followed by a TCF bleaching sequence. This allowed obtaining cellulosic pulp, hemicelluloses, and lignin as main compounds with innovative green processes.

Material and methods

Materials

Monterey pine (Pinus radiata D. Don) was obtained from the forest areas of the Basque Country (Spain). Wood was debarked, sawed, stored, and conditioned before the heat-treatment at Torresar® Company in fulfillment of the chain of custody according to the PEFC certification. The boards of Pinus radiata were principally from heartwood with the annular rings at 30 degrees to the face of the board (tangential grain). The load of Pinus radiata was modified in an industrial chamber according to industrial production standards at 212 °C [START_REF] Bruno | Artificial weathering of heat-treated pines from the Iberian peninsula :: BioResources[END_REF] under a saturated steam atmosphere for 60 h (Termogenik®, Spain). The modification process begins with a rapid increase of the temperature to 100 °C, allowing the drying of the wood to 3-4% of moisture content. Subsequently, steam is sprinkled to avoid wood damage, and the temperature in the chamber is raised to its maximum temperature (212 °C). The last stage is the cooling down and stabilizing of the samples. This stage takes about 24 h, at controlled relative humidity until room temperature to avoid abrupt temperature and pressure fluctuations (Herrera et al., 2016b).

A set of samples (20 dried wood and 20 thermally modified wood) with the dimensions of 9 x 170 x 170 mm were exposed twelve months to natural weathering in wooden support at 45 degrees of inclination and facing south direction (from May to April) in a location with oceanic climate (San Sebastian, Spain: 43°18'33.054" Latitude N; 2°0'34.817" Length W). Subsequently, weathered samples were conditioned at 20 °C and 65% RH before analysis reported previously [START_REF] Bruno | Artificial weathering of heat-treated pines from the Iberian peninsula :: BioResources[END_REF]. Samples of dry Monterey pine (Po), weathered dry pine (Pw), thermally treated pine (To), and weathered thermally treated pine (Tw) were statistically selected to compare the influence of the weathering process on the lignocellulosic components of pine and thermally treated pine. In detail, a multiple comparison procedure analysis of variance (ANOVA) was used to determine which means were significantly different from others, and the confidence levels were examined. Bonferroni Significant Difference (BSD) was applied after rejecting the null hypothesis. The software used for this statistical and graphing analysis was OriginPro 9.7.0.185.

Fractionation processes

The stages of the fractioning were selected to develop a globally environmentally friendly process to leverage all the lignocellulosic platforms as different biomaterials from this forestry product after the end-life, as reported by this group (Fernández-Rodríguez et al., 2017b;[START_REF] Robles | Production of cellulose nanoparticles from blue agave waste treated with environmentally friendly processes[END_REF]. For this purpose, acid pretreatment, organosolv treatment, and totally chlorine-free bleaching were selected. These stages constitute a sustainable process, focusing on the low environmental impact of the chemicals used through all the involved steps. All the reactions were carried out in a 4 L Zipperclave (Autoclave Engineers, Division of Snap-tite, Inc.) with PCcontrolled stirring, pressure, and temperature. The global flowsheet of the followed process and the description of the obtained samples are detailed in Figure 1.

Figure 1. Flowsheet of the process and the recovered fractions.

Acid pretreatment (AP)

All samples were chipped to an average of 10 mm chips and washed with a 0.1 M oxalic acid solution for 2 h at 100 °C [START_REF] Barana | Biorefinery process for the simultaneous recovery of lignin, hemicelluloses, cellulose nanocrystals and silica from rice husk and Arundo donax[END_REF]. Hemicelluloses were precipitated from the liquid fraction with 4 volumes of ethanol at 3 °C for 24 h [START_REF] Reyes | Extraction and characterization of hemicelluloses from Pinus radiata and its feasibility for bioethanol production[END_REF], after which the solid precipitate was separated by centrifuging the suspension and washing the pellet until neutral pH was achieved; ethanol used in this stage was recovered via rotary evaporation and used for the following stage. The recovered solid fraction was called H1; an aliquot of the liquid after centrifuging was kept to analyze non-precipitated sugars.

Organosolv delignification (OT)

The organosolv process was conducted using a mixture of ethanol/water (65:35 v/v) as a solvent, at 200 °C for 90 min and fiber to liquid ratio of 1:10 (w/v), adding 0.05M MgSO4 as catalyst [START_REF] Ni | Review: Using magnesium hydroxide as the alkali source for peroxide bleaching of mechanical pulps-process chemistry and industrial implementation[END_REF]. The liquid fraction was separated via filtration, and the solid fraction was washed several times until driving the remaining fibers to a neutral pH. Lignin (L1) was precipitated from the spent liquor by adding two volumes of acidified water (pH = 2), after which the solid fraction was filtered with a 0.22 µm membrane. Hemicelluloses that could be extracted during this process were isolated by the remaining filtrate concentration, adding 4 volumes of ethanol, and kept at 3 °C for 24 h; afterward, the solid precipitate (H2) was isolated by centrifugation.

Bleaching sequence

The Totally Chlorine Free Bleaching sequence was designed to purify the fibers obtained during the delignification stage. This sequence was based on two alkaline oxygen stages (1O2 & 2O2); one stage hydrogen peroxide with a secondary chelating reaction (PQ) and the final one with alkaline peroxide under oxygen atmosphere (PO). Oxygen-alkaline stages (1O2 & 2O2) were equally carried out with 1.5 wt% NaOH for 60 min at 98 °C, and the pressure of the autoclave was maintained at 6 bar using O2 atmosphere [START_REF] Ibarra | Integrating laccase-mediator treatment into an industrial-type sequence for totally chlorine-free bleaching of eucalypt kraft pulp[END_REF]. The liquid waste fractions from these stages were collected to precipitate lignin by their acidification using H2SO4 (96 wt%) until pH = 2. In this sense, two more lignin fractions were obtained (L2 and L3). The first stage with hydrogen peroxide was done using 3M H2O2, at pH=11, stabilized with a mixture of NaOH and Mg(OH)2 (3:1 wt%). The use of Mg(OH)2 as a partial substituent of NaOH was effectuated as it has been reported its efficiency as an alkaline agent for peroxide bleaching with a 30% substitution being the optimal [START_REF] Kong | A partial magnesium hydroxide substitution for sodium hydroxide in peroxide bleaching of an aspen CTMP[END_REF]. The reaction was performed for 120 min at 105 °C with N, N-bis(carboxymethyl)glutamic acid (GLDA, CAS: 58976-65-1) as a chelating agent. GLDA constitutes an environmentally friendly substitution of the traditional chelating agents, such as EDTA or DTPA, which are difficult to be eliminated from water effluents [START_REF] Koodynska | Chelating Agents of a New Generation as an Alternative to Conventional Chelators for Heavy Metal Ions Removal from Different Waste Waters[END_REF]. The final stage was performed using at 105 °C for 150 min; the reaction was kept under constant pressure of 6 bar of an O2 atmosphere [START_REF] Robles | Production of cellulose nanoparticles from blue agave waste treated with environmentally friendly processes[END_REF]. In this stage, a 3M H2O2 was used, the pH was stabilized at pH=11 with NaOH, without any chelating agent. For all the reactions, the liquid to solid ratio was 10:1 (w/v).

Analytical methods

The initial characterization of the raw material was carried out according to standard methods developed by the Technical Association of the Pulp and Paper Industry (TAPPI): moisture (TAPPI T 264 cm-07, 2007), inorganic content (TAPPI T 211 om-16, 2016), ethanol-toluene extractives (TAPPI T 204 cm-07, 2007) Klason lignin (TAPPI T 222 om-11, 2011), holocellulose [START_REF] Wise | Chlorite holocellulose, its fractionation and bearing on summative wood analysis and on studies on the hemicelluloses[END_REF], cellulose, and hemicelluloses [START_REF] Pettersen | The Chemistry of Solid Wood[END_REF] were quantified. Scanning electron microscopy images were obtained with a Scanning electron microscope JEOL JSM-6400 F with field emission cathode and a lateral resolution of 10-11 Å at 20 kV. Samples were analyzed by Fourier Transform Infrared Spectroscopy (FT-IR) with a PerkinElmer Spectrum Two Spectrometer, equipped with a universal Attenuated Total Reflectance (ATR) accessory with an internal reflection diamond lens. The defined range of wavelength was from 4000 to 400 cm -1 and the resolution 4 cm -1 . For each sample, 30 scans were recorded.

The cellulose fraction was analyzed by X-ray diffraction with a Panalytical Phillips X'Pert PRO multipurpose diffractometer. Samples were mounted on a zero background silicon wafer fixed in a generic sample holder, using monochromatic CuKα radiation (λ = 1.5418 Å) in a 2θ range from 5 to 50 with a step size of 0.026 and 80 s per step at room temperature. The NMR spectrometry for 13 C was performed using a Bruker 500 MHz spectrometer (Billerica, MA, USA) at 250 MHz of frequency at room temperature. The spectrum was recorded with cross-polarization and magic-angle spinning.

The samples from the stages where the hemicelluloses were precipitated, namely H1 and H2, were analyzed via high-performance liquid chromatography (HPLC). This analysis was performed using a Jasco LC Net II/ADC chromatograph equipped with a refractive index detector and a diode array detector to determine the concentration of the main monomeric sugars (glucose, xylose, arabinose, and mannose) and degradation products (acetic acid and formic acid). These compounds were analyzed using a column Phenomenex Rezex ROA. The mobile phase was a solution of 0.005N H2SO4 with a flow rate of 0.35 mL min -1 and a column temperature of 40 °C. For this analysis, the solid samples of H1 and H2 were previously dissolved in acidified water, and then they were filtered through membranes of 0.22 µm pore size. For the quantification of the main monomeric sugars and degradation products, standards of high purity and a known concentration of D-(+)glucose, D-(+)-xylose, D-(-)-arabinose, and D-(+)-mannose, acetic acid glacial and formic acid were employed.

Lignin was evaluated in terms of purity and composition, measuring the Acid Insoluble Lignin (AIL), as well as Acid Soluble Lignin (ASL) following standard methods for quantifying the purity of sulfur-free lignin samples (TAPPI T 222 om-11, 2011;TAPPI T 249 cm-09, 2009). Carbohydrates extracted during the AIL method were also determined by High-Performance Liquid Chromatography (HPLC), following the same method described above.

The molecular weight average (Mw) and polydispersity index (Mw Mn -1 ) were measured by Gel Permeation Chromatography (GPC) using a Jasco LC-Net II/ADC device, equipped with a reflex index detector, PolarGel-M column (300 mm, 7.5 mm), and PolarGel-M guard (50 mm 7.5 mm). Samples were prepared in dimethylformamide with 0.1 % lithium bromide as a mobile phase, injected using a 0.7 mL min -1 flow at 40 °C.

The calibration was carried out using polystyrene standards ranging from 266 to 70,000 g mol -1 .

In addition, lignin samples were characterized by pyrolysis-gas chromatography-mass spectroscopy (Py-GC-MS), where each sample was thermally degraded in an inert atmosphere, after which its macromolecules break down at specific lower bonding energy points, forming volatile fragments that provide useful structural information about lignin as a whole. The Py-GC-MS was carried out following the method described by this group using a commercial pyrolyzer (Pyroprobe model 5150, CDS Analytical Inc., Oxford, PA) [START_REF] Herrera | Characterization of hydrothermally treated wood in relation to changes on its chemical composition and physical properties[END_REF]. A Thermogravimetric Analysis (TGA) was also carried out to determine the thermal transitions and the thermal stability of the lignins. This characterization was carried out in a TA Q2500 calorimeter, with a 10 °C min - 1 heating ramp, from 30 to 800 °C under an N2 atmosphere.

Results and discussion

Feedstock characterization

As the first step, it is shown in Figure 2 the characterization of the different feedstock used in this work. As it can be seen, during the weathering of the non-thermally treated material (Pw vs. Po), the percentages of the carbohydrate macrocomponents (celluloses and hemicelluloses) were reduced, whereas the portion of lignin increased, showing in all cases a significant difference at the p<0.05 level. This involves the weathering affected mainly the polysaccharides that are easier to be degraded (hemicelluloses and the amorphous cellulose section).

On the contrary, it was more difficult to degrade the aromatic lignin structures, which led to an increase in the lignin percentage in Pw. The use of the thermally treated sample (To vs. Po) provoked the same effect as the weathering, but more intense, with a higher decrease of the percentage of carbohydrates (especially hemicellulose, which showed a significant reduction at the p<0.05 level) and the corresponding increase of the lignin fraction. This was caused by the solubilization of the hemicelluloses and the small amorphous portion of the cellulose, remaining the lignin in a bigger concentration due to its stabilization by the polycondensation reactions of lignin undergone during the thermal treatment, as reported by Herrera and collaborators (Herrera et al., 2016a). However, a trend change was experienced when the weathering was applied over a thermally treated material (To) since the carbohydrate platform increased in percentage of the remaining material after the weathering. At the same time, the lignin content was decreased, in the opposite tendency as in Pw. The reason is based on the influence of the thermal pretreatment, as during this step, the carbohydrate composition that was more prone to be extracted was already solubilized, this resulted in no significant differences (p<0.05) between the lignin content of Pw and Tw. In this sense, the portion of carbohydrates still in the feedstock was more recalcitrant to be degraded by the exposure to the environment. Therefore, the removal of some carbohydrates during the thermal pretreatment led to generating a substrate more resistance and steady during the weathering exposure, as the weaker parts of the carbohydrate platform were already extracted, which can be considered a strong advantage of the thermal treatment. Nevertheless, the lignin composition increase led to higher availability, comparing To against Po, to be attacked by the weather conditions, since lignin is a sensitive component to be photo-degraded by weathering [START_REF] George | Photodegradation and photostabilisation of wood -the state of the art[END_REF]. Figure 2. Chemical composition of the used biomass (Po: Monterey pine; Pw: weathered pine; To: treated pine; Tw: weathered thermally treated pine), further information is presented in Table S7.

Fractionation yields

The relative extraction yields for each recovered fraction (cellulose, lignin, and hemicelluloses) were presented in Figure 3. Tables S1 to S6 present additional information concerning the recovered fractions after each process.

The extraction yield of cellulose (C1), expressed as the cellulose content of the pulp obtained after the whole process, showed similar yields for the materials that were not thermally treated (Po and Pw). However, there was a decrease in the cellulose yield in the case of To (45.46%). Therefore, the thermal treatment provoked a decrease in cellulose in the initial material, as shown in Figure 2. This different behavior of To was not experienced by Tw, as the extraction yield (61.11%) was closer to the Ry of Po (65.9%) and Pw (64.07%). Thus, in this case, the weathering mitigated the influence of the thermal treatment.

From the lignin yields, it can be established that the process to extract lignin by this route was adequately designed as it allowed almost a total Ry for the Po, with a total lignin extraction (L1+L2+L3) of around 98.62%. However, when the initial material was subjected to thermal treatment or a weathering period, the extraction of lignin in the OT stage was deeply impacted, with a reduction of L1 from ≈65% in Po to 30-40% for the other feedstock. Regarding the yields from different stages, the main extraction occurred during the organosolv process (L1) as was expected, especially in non-thermally treated pine (Po and Pw). Although the L1 samples from the thermo-treated wood (To and Tw) showed lower extraction yields for L1, the yields for L2 (31.15% and 24.33%, respectively) were similar to L2 extraction yields for Po and Pw (31.35% and 13.18%). The reason can be based on the new nature of the remaining lignin or para-lignin after the thermal treatment, as reported before [START_REF] Herrera | Characterization of hydrothermally treated wood in relation to changes on its chemical composition and physical properties[END_REF], where it was pointed out that the original lignin undergoes several reactions of polycondensation above 190 °C. These reactions are caused by the initial demethoxylation of the methoxy groups of guaiacyl and syringyl units that allowed free ortho sites in the aromatic rings to be cross-linked with other components fragments of cellulose and hemicelluloses when the temperature was above 190 °C. This more condensed structure is less sensitive to be extracted by the solubilisation with organic solvents (OT), whereas it is possible to be degraded and extracted by the alkaline treatment applied during the 1O2 and 2O2 stages. Therefore, it was justified the inclusion of the lignin precipitation stage after the 1O1 stage. On the contrary, the fraction of L3 recovered after the 2O2 stage was inefficient, based on the low extraction yields (0.5-2.5% for all the materials). Thus, this fraction could be considered almost negligible.

Hemicelluloses recovery resulted in low yields regardless of the feedstock. This is caused because for hemicelluloses (H1, H2, and H3), only solid fractions were considered, not the small fractions (sugars and acids) that were not capable of being precipitated by the method described before. In any case, the extraction stages (AP y OT) were intentionally designed using mild conditions to allow the extraction of the hemicelluloses without degrading them into their monomeric compounds. H1 and H2 were the precipitated fractions from AP and OT, whereas the H3 was comprised in the solid fraction in the final recovered pulp after the bleaching sequence. This is why H3 obtained a higher recovery yield since only the bigger molecular compounds of the extracted hemicelluloses could be isolated by this precipitation method. While this technique could not recover the monomeric sugars and small oligomers, which were neglected due to the low concentration, it was quantified in the liquid streams and their difficulty to be valorized in value-added applications. This is due to their lack of biocompatibility based on the pH of the extracted liquid streams, despite the approach followed in other works [START_REF] Gullón | Valorization of by-products from olive oil industry and added-value applications for innovative functional foods[END_REF]. Consequently, the most remarkable yield was reached by To, because the remaining hemicelluloses after the thermal treatment are more condensed and more accessible to be isolated by their precipitation, despite their lower composition in the feedstock.

Regarding process efficiencies (the total amount of solids recovered after fractionation), they were similar during the first two processes: the process efficiency of Organosolv 84.18% and the first alkaline extraction 85.19 % (tables S2 and S4 respectively of supporting information). During the second alkaline extraction, the efficiency was almost 100%, while the recovered lignin was, in every case, less than 1 gl gm -1 ; the recovered solid fraction was practically not changed. This can be related to the process reaching its inherent feasibility to extract lignin, a phenomenon observed in previous work from De Hoyos-Martínez and collaborators (de Hoyos-Martínez et al., 2018), and less pronouncedly in the work of Fernández-Rodríguez and collaborators (Fernández-Rodríguez et al., 2017b), in which sequential treatments of similar nature resulted in considerably lower yields. Process efficiencies of hydrogen peroxide stages (P) correspond to the recovered bleached pulp, as no other product could be recovered; in the case of the first stage (PQ), the mean process efficiency was 75.51%, being 83% for pinewood and 72% for thermowood. It can be appreciated that the total lignin recovered corresponded to a total of 98.63 % for Po and 61.00% for To without weathering, while the recovery of lignin after weathering was 51.19% for Pw and 58.94% for Tw. This implies that the lignin recalcitrance for extraction with the Organosolv and 1O2 bleaching stage does not change after weathering for T samples, while for P, it has an important decrease in the effectiveness during the 1O2 stage. Figure 3. Yields of each recovered solid fraction concerning the initial macrocomponent amount reported in Figure 2. C1 is the cellulose content of the bleached pulp. L1 is the organosolv lignin, L2 is the alkaline lignin from bleaching sequence O1, L3 is the lignin from bleaching sequence O2. H1 is the hemicelluloses from acid pretreatment, H2 are the hemicelluloses from organosolv, and H3 are hemicelluloses remaining in the bleached pulp.

Physico-chemical characterization of the obtained products 3.3.1. Cellulose fraction

Infrared spectrometry analysis was performed on the cellulose fractions recovered to evaluate their structure (Figure 4). Infrared spectra of bleached pulp (C1) show a broad band at 3600-3000 cm -1 , attributed to free vibrating hydroxyl groups characteristic of cellulose. Bands corresponding to v(C-H) and v(CH2) locate between 3000-2800 cm -1 displayed a weak intensity before thermal treatment of wood; in the case of To and Tw, they are almost imperceptible. Regarding the fingerprint region, it can be identified a band at 1430 cm -1 related to Methoxyl-O-CH3 bonds and a band at 1370 cm -1 corresponding to C-H bond in -O(C=O)CH2 group. Bands at 1317 and 1200 cm -1 are attributed to -OH groups in-plane bending, while the band at 1160 cm -1 corresponds to the C-O-C anti-symmetric bridge stretching in cellulose. Below 1200 cm -1 , there can be identified two bands at 1100 and 1055 cm -1 corresponding to C-OH and C-O stretching, and finally, a band at 900 cm -1 that corresponds to the β-glucosidic linkages between sugar units [START_REF] Kruer-Zerhusen | Characterization of cellulose crystallinity after enzymatic treatment using Fourier transform infrared spectroscopy (FTIR)[END_REF]. In this fingerprint region, there is no particular difference between the analyzed celluloses, which implies that the chemical structure of cellulose is not significantly affected by either the thermal treatment or the effects of weathering. Figure 4. Infrared absorbance spectrograms of the obtained celluloses.

Figure 5 presents the 13 C CP-MAS NMR analysis of the obtained bleached pulps, composed mainly of cellulose.

Insets present a magnification of the C4 region, which is considered a referent to identify the more ordered structures of cellulose I. NMR analysis shows that the structure of pulps was practically invariable despite weathering. This consistency was a positive outcome if weathered thermally treated wood would be considered as raw material for the pulp and paper industry during a cascading use of such material. The main difference observed was between Po and Pw, with an increase of the amorphous region of the C4 chemical shift, while for To and Tw, the change in proportions was inverse, with Tw having a sharper shift at the ordered region (92-86 ppm). This change can be related to the transformation of low molecular carbohydrates during treatment, which reacted with lignin and leached during weathering. This resulted in a pulp enriched in more crystalline and ordered cellulose, which is a phenomenon already reported to happen in thermally treated wood regardless its origin (softwood or hardwood), previous authors have reported maximal crystallinity being reached around 210 °C, similar to the conditions used in this work [START_REF] Durmaz | Effects of Heat Treatment on Some Characteristics of Scots Pine (Pinus sylvestris L.) Wood[END_REF][START_REF] Yildiz | The effects of thermal modification on crystalline structure of cellulose in soft and hardwood[END_REF].

Figure 5. NMR spectra of the obtained celluloses

The x-ray diffraction patterns of the bleached pulps are presented in Figure 6. Three main components can be observed, cellulose Iβ, which was the major component, as it is typical for native celluloses, cellulose Iα in a minor proportion, and traces from Mg(OH)2, which were residuals from the first alkaline-oxygen bleaching stage.

As calculated from the XRD patterns with the peak fitting method and subtracting the contribution made by Mg(OH)2, crystallinities were in general high, with Po having 70.4%, Pw 68.25%, To73.69, and Tw 71.48%. This means that crystallinity was slightly increased during thermal treatment, mainly due to interaction between amorphous cellulose with lignins to form lignin-carbohydrates complexes (LCC), but it was also reduced after weathering because of cellulose degradation. It can be concluded that there was a direct relationship between the crystallinity of the cellulose and its affinity to keep Mg(OH)2 attached to the pulp when comparing results

from Figures 5 and6. Po and Tw were the ones having more intense signals corresponding to the Mg(OH)2 [START_REF] Maskal | Magnesium hydroxide-containing paper[END_REF][START_REF] Ni | Review: Using magnesium hydroxide as the alkali source for peroxide bleaching of mechanical pulps-process chemistry and industrial implementation[END_REF]. Figure 6. X-ray diffraction patterns of the different celluloses.

Hemicellulose fraction

Pure hemicelluloses streams were recovered from two different stages of the global process; leaching (H1) and organosolv (H2), as H3 was contained within the final pulp stream. Their monomeric composition characterized these precipitates both in sugars and in acids, whose quantified amounts are presented in Table 1. The most common sugar was mannose-related oligosaccharides, as expected from softwood [START_REF] Santos | Effect of autohydrolysis on Pinus radiata wood for hemicellulose extraction[END_REF]. The presence of mannose in H1 was affected by two parameters. First, the thermal treatment, showing a decrease of ≈37% in To compared to Po, and the second was the weathering, having Pw a decrease of ≈23% compared to Po. When both parameters were present (Tw), the decrease in mannose concentration was ≈51%. This means that, while the cellulose was preserved almost identical through the treatment and weathering, the oligosaccharides present in the pinewood were more severely affected by these two parameters, with xylose concentrations and arabinose oligosaccharides suffering similar tendencies in the case of H1. On the other hand, H2 samples were more similar; however, the overall purity was strongly diminished; this is due to the severity of the treatment, but also because of the difficulty to recover dissolved polysaccharides from organosolv liquors, given the fact that hemicelluloses precipitate in organic solvents [START_REF] Peng | Fractional purification and bioconversion of hemicelluloses[END_REF]. Table 1. The concentration of monomeric sugars and acids in the recovered products (expressed in mg L -1 The infrared spectra of hemicelluloses obtained from the acid leaching (H1) presented a broad band at 3600-molecules. A band at 2000-2700 cm -1 corresponded to -CH bond deformation of -CH2 and -CH3, these bands were absent in To and Tw, meaning that most of the hemicelluloses were depolymerized during the hydrothermal treatment [START_REF] Boonstra | Chemical analysis of heat treated softwoods[END_REF]. The spectra of H1 from To and Tw were more similar to xylose, showing weaker bands attributed to C-H vibrations of mannose (1500-1200 cm -1 ) than Po and Pw, and weak peaks associated with xylose (1200-900 cm -1 ), thus showing xylose degradation [START_REF] Wang | Catalytic pyrolysis of mannose as a model compound of hemicellulose over zeolites[END_REF]. Hemicelluloses obtained from the Organosolv treatment (H2) presented fewer differences between each other. Thus, increasing the severity of the treatment, the obtained samples were more homogeneous regardless of the used feedstock in the process. In H2, it can be highlighted the identifiable bands corresponding to hydroxyl groups (3600-3200 cm -1 ), the band at 1650 cm -1 corresponds to CO stretching of carboxylate in either salt or ester form (Z. Chen et al., 2015). Finally, characteristic C-O-C stretching of the pyranoid ring of xylan was observed at 1060 cm -1 , and the band corresponding to β-glucosidic linkages between xylose units is present at 890 cm -1 [START_REF] Sun | Hemicellulose-based pH-sensitive and biodegradable hydrogel for controlled drug delivery[END_REF]. Results from infrared spectra were concurrent with the concentration analysis, showing a reduction in mannose concentration for H1 in To and Tw compared with Po and Pw; while for H2, the spectra presented lower variations. Figure 7. Infrared absorbance spectrograms of the obtained hemicelluloses from acid pretreatment (H1) and organosolv (H2).

Lignin fraction

Table 2 presents values corresponding to acid-insoluble lignin (AIL), acid-soluble lignin (ASL), residual carbohydrate contents, ashes, molecular weight (Mw), and polydispersity index (Mw Mn -1 ) of the obtained lignin samples. The aggregate of ASL and AIL is related to the purity of the obtained lignin, in which it can be observed a high purity of the organosolv lignins (L1) with no significant differences between the samples extracted from the different feedstock (93-95% for all of them). This means that despite the differences between yields, the properties of lignins obtained by the OT stage shared the same composition. Besides, the higher purity of the isolated lignin, compared to other results presented in the literature [START_REF] Hosseinaei | Role of Physicochemical Structure of Organosolv Hardwood and Herbaceous Lignins on Carbon Fiber Performance[END_REF][START_REF] Toledano | Fractionation of Organosolv Lignin from Olive Tree Clippings and its Valorization to Simple Phenolic Compounds[END_REF], could be based on introducing the pretreatment stage, where the carbohydrate compounds that could pollute the lignin samples were significantly reduced by the Ap stage; in line with other similar works previously carried out by this research group (Fernández-Rodríguez et al., 2017a). This was also countersigned by the low presence of sugars in the collected samples, with values between 1.70 and 1.85% except for Tw lignin, in which the carbohydrate content of the lignin samples was above 2.5%. This increase can be related to the recalcitrance caused by the LCC formed during the thermal treatment, which were not leached during weathering neither hydrolyzed during the AP treatment. This tendency was also observed in alkali lignins from the first bleaching step (L2), in which both weathered samples (Pw and Tw) presented higher carbohydrate content than their original counterparts, following the same trend already reported in previous work (Fernández-Rodríguez et al., 2017b). In any case, the purity of the L2 samples was above 90% (except in the case of Po), demonstrating once again that the inclusion of a lignin isolation step could be implemented even during the first bleaching stage since the purity of the lignin was not compromised. Nevertheless, the lignin precipitation from the 2O2 stage was not justified due to the low amount obtained, which was not big enough to obtain the necessary material even to conduct its full characterization. Figure S2 shows a graphical display to explain better the occurring tendencies regarding molecular weight and polydispersity index. In the untreated pinewood, the molecular weight of lignins after weathering was higher in all cases (L1, L2, and L3), presenting an almost linear trend and, therefore, a correlation between the Mw of each lignin type. The Mw of organosolv lignin (L1) in all samples was decreasing after thermal treatment, as it is represented in Figure S3. On the contrary, the Mw of alkali lignin (L2) increased after the thermal treatment of the samples. andL3) were subjected to infrared analysis and carbohydrate products. Besides having different yields depending on the stage from which they were recovered, there were noticeable differences between the three of them according to their functional groups, with more differences found depending on the source. Absorption band associated with -OH stretching vibrations of aromatic and aliphatic groups between 3700 and 2900 cm -1 presented a distinct difference for L1. On the other hand, for L2, the intensity of that absorbance band was lower in the case of To and Tw compared to Po and Pw, and in the case of L3, the presence of the OH band was almost imperceptible. The bands between 2900 and 2750 cm -1 , which correspond to CH stretching in -CH2 and -CH3, were more intense in the case of bleaching-stage lignins (L2 and L3) than in Organosolv lignin (L1). Moreover, in L1, the C-H band was more acute for To and Tw, which correlated with the absence of such bands in H1. This phenomenon can be related to the formation of LCC during hydrothermal treatment. The band at 1710 cm -1 , identified with non-conjugated carboxylic acids, was present in all lignins, with a tendency to decrease after each treatment (L1>L2>L3). Another particularity was the increase of the intensity of this band for To and Tw in the case of L1, which can be related to the degradation of small carbohydrate sugars into carboxylic acids. The band between 1700 and 1400 cm -1 was related to the aromatic skeletal vibrations of lignin. In this region, the same decreasing tendency can be observed, which implies a lower quality of lignin found during the cascading recovery. In this region can be highlighted the band at 1595 and 1510 cm -1 , which corresponds to C=C linkages of the skeletal aromatic ring, the band at 1460 cm -1 corresponding to C-H deformation, and the band at 1420 cm -1 corresponding to C-H aromatic ring vibrations. In the region between 1500 and 1000 cm -1 , the prominent guaiacyl lignin bands can be identified, namely 1270, 1120, and 1030 cm -1 , which are related to v (C-O stretching) of the guaiacyl ring, v(C-H stretching) of the guaiacyl unit, and v(C-O and C-H) in-plane deformation of G-type lignin respectively [START_REF] Derkacheva | Investigation of Lignins by FTIR Spectroscopy[END_REF]Fernández-Rodríguez et al., 2017b;[START_REF] Ibarra | Structural modification of eucalypt pulp lignin in a totally chlorine-free bleaching sequence including a laccase-mediator stage[END_REF][START_REF] Lu | Structural Characterization of Lignin and Its Degradation Products with Spectroscopic Methods[END_REF][START_REF] Xu | Qualitative and quantitative analysis of lignocellulosic biomass using infrared techniques : A mini-review[END_REF]. These bands presented a gradual decrease after each treatment, with the first alkali extracted lignin (L2) from hydrothermally treated wood (To and Tw) having the weakest absorbance intensity in this region. The thermal properties of the different lignin samples were subjected to a thermogravimetric (TG) analysis. Figure 9 presents the thermograms of L1 and L2 lignins under an inert atmosphere; insets show the derivative thermogram. Other parameters, such as the temperatures of 5% mass loss, 50% mass loss, and residues after 800 °C, are represented in Figure S4. Regarding the amount of the remaining residue, the main difference was appreciable between the final residue for L2 compared to L1, as L2 residues are between 42.39% (Po) and 51.09% (To), while L1 residues are between 33.36 (Po) and 39.74 (Tw). This situation was related to the origin of the lignin since more condensed structures were expected from the 1O2 stage against L1 from OT. Besides, it can be highlighted that there was a clear tendency to increase the residue at 800 °C for thermally treated wood, regardless the stage the lignin samples were isolated (L1 or L2). This can be based on the more stable lignin is conformed during the thermal treatment, as it was commented before. The TG analysis under inert atmosphere gives information regarding the potential char production for each lignin; therefore, a higher residue was related to a higher char content. The differences in decay temperatures and the thermal stability of lignins are related to their structure, molecular weight, origin, and the extraction and precipitation methods followed to obtain the lignins [START_REF] Gordobil | Assesment of technical lignins for uses in biofuels and biomaterials: Structure-related properties, proximate analysis and chemical modification[END_REF]. In this sense, L1 presented higher thermal resistance, presenting the maximum degradation temperature at higher temperatures than those of L2. It can also be seen that To and Tw had higher thermal resistance than Po and Pw. Moreover, Pw and Tw had higher maximum degradation temperatures than Po and To, respectively. On the other hand, L2 lignins presented lower differences between Po and To, being Pw, the one with the highest thermal maximum degradation temperature (389 °C), lower than all of the L1 lignins. From the TG analysis, it can be concluded that L2 lignins were more prone to produce char than L1 samples. This can be due to the OT stage being more effective in extracting less altered lignin structures. Simultaneously, the oxidizing effect of oxygen, hydrogen peroxide, and the alkaline media influence the denaturalization of these compounds, resulting in lignins with more condensed structures. To conclude the lignin characterization, the structural composition of the lignin samples was analyzed by the Py-GC/MS technique. The analysis is carried out based on the work developed by Chen and collaborators (L. Chen et al., 2015). The identified phenolic derivatives were classified into five categories, namely benzene (B), phenol (P), catechol (C), guaiacol (G), and syringol (S) derived units. As it is not clear that catechol could be originated from G or S structures, the results were also expressed as the S/G ratio to facilitate the interpretation of the results. Guaiacol was the main compound identified by this technique, a typical fact in lignin samples obtained from softwood species. Thus, the S/G ratio was always close to zero. The highest values were obtained by Po lignin samples regardless of the stage they were isolated from (L1 and L2). This indicates that both the thermal treatment and the weathering attack to a more prominent degree the S units, since the natural weathering of wood (especially UV radiation) and thermal treatment could promote demethoxylation reactions that resulted in the transformation of S derived units into C and G derived moieties. This tendency has already been reported [START_REF] Ma | Study on the thermal degradation behaviors and kinetics of alkali lignin for production of phenolic-rich bio-oil using TGA-FTIR and Py-GC/MS[END_REF]. The G units lead to creating more condensed structures due to their availability of the C5 in the aromatic ring. Lignin samples from To feedstock show the second highest values; thus, weathering causes this degradation mechanism to a higher degree than the thermal treatment. Among samples from different stages, the differences were not significant, although there was a trend to reduce the S/G in L2 samples in comparison with L1, a fact that can be explained for the mechanisms described before since, during the 1O2 stage, more demethoxylation reactions of the S units could appear. Moreover, they followed a similar decreasing tendency in both organosolv and alkaline lignins after the weathering treatment. Table 3. The abundance of the different phenolic-derived units in the lignins respects the total phenolic moieties detected.

Benzene

Conclusions

The present work proposed a cascade valorization of thermally treated wood after service-life by fractioning its macromolecular components through green processes. To assess the quality of the obtained materials, they were compared with untreated wood and unweathered counterparts. Results showed that the macromolecular constituent that presented the most stability regardless of the conditions was cellulose, having little structural properties changes regardless of the feedstock used as raw material, which demonstrated the robustness of the designed process as the main product in yield was very homogeneous, a fact that would enable the scalability of this global process. The extracted hemicelluloses consisted of high purity mannose oligomers; however, due to the difficulties of precipitating hemicelluloses in its polymer nature, the use of the organosolv process and the acid leaching were not enough to justify a valorization of hemicelluloses, as their low extraction yields demonstrated it. Lignins were the macromolecules presenting more variations, with significant differences depending on the extraction phase (organosolv pulping or oxygen-alkali bleaching) and the material source. With thermally treated wood having more LCC (oxygen-alkali lignin) but also the best yield of AIL (organosolv lignin), however, in general, the purity and the thermal stability of all the lignins resulted in high values for either lignin, stating that the selected processes are robust and appropriate for the fractioning of pinewood after being thermally treated and weathered.

Supporting Information.

Please consult the supplementary information for additional data.

Figure 8 .

 8 Figure 8. Infrared absorbance spectrograms of the obtained lignins.The thermal properties of the different lignin samples were subjected to a thermogravimetric (TG) analysis. Figure9presents the thermograms of L1 and L2 lignins under an inert atmosphere; insets show the derivative thermogram. Other parameters, such as the temperatures of 5% mass loss, 50% mass loss, and residues after 800 °C, are represented in FigureS4. Regarding the amount of the remaining residue, the main difference was appreciable between the final residue for L2 compared to L1, as L2 residues are between 42.39% (Po) and 51.09% (To), while L1 residues are between 33.36 (Po) and 39.74 (Tw). This situation was related to the origin of the lignin since more condensed structures were expected from the 1O2 stage against L1 from OT. Besides, it can be highlighted that there was a clear tendency to increase the residue at 800 °C for thermally treated wood, regardless the stage the lignin samples were isolated (L1 or L2). This can be based on the more stable lignin is conformed during the thermal treatment, as it was commented before. The TG analysis under inert atmosphere gives information regarding the potential char production for each lignin; therefore, a higher residue was related to a higher char content. The differences in decay temperatures and the thermal stability of lignins are related to their structure, molecular weight, origin, and the extraction and precipitation methods followed to obtain the lignins[START_REF] Gordobil | Assesment of technical lignins for uses in biofuels and biomaterials: Structure-related properties, proximate analysis and chemical modification[END_REF]. In this sense, L1 presented higher thermal resistance, presenting the maximum degradation temperature at higher temperatures than those of L2. It can also be seen that To and Tw had higher thermal resistance than Po and Pw. Moreover, Pw and Tw had higher maximum degradation temperatures than Po and To, respectively. On the other hand, L2 lignins presented lower differences between Po and To, being Pw, the one with the highest thermal maximum degradation temperature (389 °C), lower than all of the L1 lignins. From the TG analysis, it can be concluded that L2 lignins were more prone to produce char than L1 samples. This can be due to the OT stage being more effective in extracting less altered lignin structures. Simultaneously, the oxidizing effect of oxygen, hydrogen peroxide, and the alkaline media influence the denaturalization of these compounds, resulting in lignins with more condensed structures.

Figure 9 .

 9 Figure 9. Thermograms and derivative weight loss as a function of the temperature of L1 and L2 of the different samples.

Table 2 .

 2 Composition of the lignin samples collected along the different stages of the experimental procedure.

			AIL [%]	ASL [%]	Carbohydrates [%]	Ashes [%]	Mw [g mol -1 ]	Mw Mn -1
		L1	93.4	0.88	1.84	1.13	5534	4.43
	Po	L2	83.3	1.46	3.01	1.15	2772	2.94
		L3	-	-	-	-	21,936	12.4
		L1	93.9	0.53	1.83	0.29	7401	5.32
	Pw	L2	93.1	1.20	4.44	0.65	10,049	8.68
		L3	-	-	-	-	28,573	9.27
		L1	93.4	0.74	1.73	0.51	1453	2.36
	To	L2	92.8	1.08	5.27	1.06	3477	4.16
		L3	-	-	-	-	23,380	12.8
		L1	94.6	0.95	2.58	0.25	4307	3.71
	Tw	L2	89.9	1.29	6.11	0.49	3465	3.88
		L3	-	-	-	-	19,233	10.3
	All the lignin samples (L1, L2,

Acknowledgments

The authors would like to acknowledge the University of the Basque Country UPV/EHU for financially supporting this work. E.R. wants to acknowledge the tenure track position "Bois: Biobased materials" part of E2S UPPA supported by the "Investissements d'Avenir" French program managed by ANR (ANR-16-IDEX-0002). R.H. wants to acknowledge the Basque Government for financial support through the INGVTCL4-D00112-7 contract and European Commission for funding the InnoRenew project (Grant Agreement # 739574) under the Horizon2020 Widespread-Teaming program, the Republic of Slovenia (investment funding from the Republic of Slovenia and the European Union European Regional Development Fund), and infrastructural ARRS program IO-0035. The authors thank for technical and human support provided by SGIker of UPV/EHU for XRD characterizations. References