PhD Maud Pétrault
email: maud.petrault@univ-lille.fr

Thavarak Ouk

Michèle Bastide

Régis Bordet

Olivier Pétrault

Vincent Bérézowski

Age and diet modify acute microhemorrhage outcome in the mouse brain

Keywords: Cerebral microhemorrhages, cognitive decline, age, high-fat diet, mouse

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

Introduction

Cognitive decline is defined as an age-related decrease in cognition. It usually becomes manifest through slight alterations of mnesic functions such as executive functions [START_REF] Tomita | Detecting cognitive decline in community-dwelling older adults using simple cognitive and motor performance tests[END_REF]. These functions deteriorate physiologically along the aging process. Still, age refers to a length of time during which humans under enriched diet can accumulate multiple metabolic risk factors for cerebrovascular disease, possibly leading to precipitated cognitive impairment. Indeed, the presence of metabolic disturbances at midlife has been linked recently to mild cognitive impairment, increasing the risk of vascular pathologies and late-life dementia [START_REF] Cuspidi | Association of metabolic syndrome with carotid thickening and plaque in the general population: A meta-analysis[END_REF][START_REF] González | Midlife cardiovascular health and 20-year cognitive decline: Atherosclerosis Risk in Communities Study results[END_REF][START_REF] Otite | Increasing prevalence of vascular risk factors in patients with stroke: A call to action[END_REF]O. Pétrault et al., 2019;[START_REF] Whitmer | Midlife cardiovascular risk factors and risk of dementia in late life[END_REF]. Among the consequences of vascular pathologies, post-stroke cognitive impairment is now well documented, but the proper effect of microlesions such as cerebral microhemorrhages (CMHs) is still a subject of debate, since confounded with the expression of concomitant brain disease in the elderly population. The presence of CMHs has yet been associated with age-related cognitive decline in the general population [START_REF] Ding | Space and location of cerebral microbleeds, cognitive decline, and dementia in the community[END_REF] and is suggested to contribute to cognitive impairment in non-demented elderly individuals [START_REF] Paradise | The relationship of cerebral microbleeds to cognition and incident dementia in non-demented older individuals[END_REF]. However, the experimental induction of CMHs has been performed in young healthy mice only and was found silent at onset but able to promote slight cognitive alterations only a midlife time after (M. Pétrault et al., 2019). Given the vascular nature of CMHs, their higher incidence or extent relates to poor vascular health as a possible result of both aging and metabolic disturbances. Middle-aged adults with metabolic disturbances might thus be subjected to potentiated effects of CMHs on brain functions at onset, or in the long term through a precipitated cognitive decline with or apart from transformation into symptomatic intracerebral hemorrhage. To test this hypothesis, we have induced a disseminated pattern of CMHs in adult mice of two different ages and times of metabolic disturbances obtained after high-fat diet (HFD, 40% fat) as previously characterized (O. Pétrault et al., 2019). We examined spontaneous locomotion, working memory and visual recognition memory, as well as the extent of the CMH lesions.

Experimental procedures

Ethical aspects

All experiments were approved by the national Ethical Committee in Animal Experimentation (CEEA n°75, Comité d'Ethique en Experimentation Animale), from the French Ministry for Education and Research (agreement number: n°2016022510527156-V2) and were performed in strict compliance with the European Union Directive 2010/63/EU. Experiments were reported in accordance with the ARRIVE guidelines for reporting experiments involving animals.

Animals and diet

One hundred and twenty-four adult male C57Bl6/J mice aged 8 weeks at the beginning of the study (Janvier Labs, Le Genest Saint Isle, France) were housed in standard cages. At the start of the study, mice were randomly assigned to either a nutritionally complete, HFD consisting of 40% saturated fat, mostly from lard (HF231, Safe, Augy, France) or normal control diet (ND) consisting of 3 % fat (A04, Safe, Augy, France) throughout the experimental period. All animals had ad libitum access to food and water. There were no significant differences in body weight between groups before the experimental diets were given, as mice had been maintained on a regular chow diet.

Induction of microhemorrhages and time points

Cerebral microhemorrhages were induced by injecting Randomly methylated-β-Cyclodextrins (RAME-CD Sigma-Aldrich, Saint-Quentin Fallavier, France), as previously described (M. Pétrault et al., 2019). Mice were anesthetized with isoflurane (induction: 3%; maintenance: 2%) and body temperature was kept constant around 37°C. The carotid arteries were exposed through a midline cervical incision and the common carotid was ligated with a silk suture.

The RAME-CD solution (200 mg/ml) was injected in the common carotid via a 29G insulin syringe (0.3g/kg). Then, the suture was carefully removed and the mouse was placed under a red warm light to recover. A maximum pattern of around 20 CMHs per brain was observed at 7 days after injection, at both cortical and subcortical levels, and used for the present study.

Three

protocols were carried out in parallel:

1. The acute effect of CMHs on locomotion and cognition of mice under metabolic disorders was tested. A short-term protocol was designed for the induction of the CMH lesions in the brains of 14-month-old mice (after 12 months of diet) or 5-month-old mice (after 3 months of diet), when metabolic, vascular, and cognitive disturbances were, respectively, well established or just beginning; this was described previously (O. Pétrault et al., 2019) (Fig. 1A and 2A). Mice were randomly divided into 2 groups (n=12-15 per group of 14 month-old mice, and n=7-8 per group of 5 month-old mice), both injected with RAME-CD and fed with ND or HFD. After 7 days, before sacrifice, mice were kept for behavioral studies in order to check for the functional impact of CMHs. Two groups of mice (n=10 per group) were inserted as controls, i.e. devoid of RAME-CD injection but fed with ND or HFD.

2. The long-term effect of CMHs on locomotion and cognition of 5-month-old mice experiencing CMHs when under early metabolic disorders (after 3 months of diet) was tested.

A long-term protocol ensured the follow-up study of the effects of CMHs in the brains of mice on ND or HFD (Fig. 4A). Mice were randomly divided into 2 groups (n=10-12 per group) fed with ND or HFD. The CMH lesions were induced after 3 months of diet and behavioral tests were performed at 6-9-and 12-month time points. At the end, mice were sacrificed for histological purposes. Two groups of mice (n=10 per group) were inserted as controls, i.e. devoid of RAME-CD injection but fed with ND or HFD.

3. A metabolic characterization (weight, blood triglycerides and total cholesterol) of each mouse was performed at 3, 6, 9 and 12 months of diet, and visceral adipose tissue was weighted after sacrifice, at 3 and 12 months, as described in (O. Pétrault et al., 2019).

Behavioral assessments

Mice of each group underwent a battery of tests evaluating spontaneous locomotor activity, visual recognition memory and working memory. These tests were chosen according to the brain areas affected by CMHs, which are those of the Sylvian territory. They were performed every morning between 9 a.m. and 2 p.m. During experiments, the equipment was cleaned with 70% ethanol after each trial so that olfactory cues did not bias the results.

-Spontaneous locomotion Spontaneous locomotor activity was assessed in an open field test device, using an infrared actimeter (Bioseb, USA). The apparatus consisted of a square arena (45 x 45 cm) with a black polymethyl methacrylate floor and transparent 34-cm-high polymethyl methacrylate walls.

Mice were placed in the center of the arena, and were allowed to explore the arena freely for 10 min. Activity was recorded by two rows of infrared photocell sensors and processed with the Actitrack software (Bioseb). The total distance covered (in cm), the duration of inactivity (resting time, in seconds) and the mean speed (cm/sec) of each mouse were measured.

-Visual recognition memory

The novel object recognition (NOR) test is based on the tendency of mice to explore a novel object rather than a familiar one. Mice were habituated to a square arena (50 x 50 x 25 cm) for 10 minutes on the first day. On day 2, mice were exposed to the arena containing two identical objects. In a first phase ("sample phase"), on day 3, mice were presented two novel and identical objects for 15 minutes. Animals returned in their cages. After one hour, they were placed back in the testing arena for 5 minutes ("test phase"), but one of the familiar object had been previously replaced by a novel object. Performance of the mice was video recorded (Ethovision XT, Noldus, Wageningen, The Netherlands). Exploratory behavior was defined as the animal directing its nose toward the object at a distance below 2 cm. Any subject that failed to complete a minimum of 20 seconds of exploration during the sample phase was excluded from the analysis. A discrimination ratio (index) was calculated as the difference between the time spent for the exploration of the novel object minus the time spent for the exploration of the familiar object divided by the sum of both [START_REF] Antunes | The novel object recognition memory: neurobiology, test procedure, and its modifications[END_REF].

-Working memory

The spontaneous alternation test was performed using a Y-maze (made of white polyvinyl chloride) and a closed-circuit video camera (Ethovision XT, Noldus, Wageningen, The Netherlands). All three arms were of the same size (30 x 8 x 15 cm) and were oriented at an angle of 120° to each other. The Y-maze was placed in a room with no environmental cues.

Each mouse was placed at the end of one arm and was allowed to move freely between the maze's three arms for 8 minutes. A visit to an arm was scored when all four of the mouse's paws were within the arm area. The sequence of the arm visits was recorded and an alternation response was scored when the animal entered the least recently visited arm. The alternation score was calculated as the ratio between actual and possible alternations (defined as the total number of arm visits minus 2), multiplied by 100 [START_REF] Hidaka | Effects of Repeated Electroconvulsive Seizures on Spontaneous Alternation Behavior and Locomotor Activity in Rats[END_REF]. This calculation was performed only for mice making more than 20 arm visits.

Determination of the CMH lesions

At the end of the protocols, mice were killed with an overdose of pentobarbital (200 mg/kg, intraperitoneal). Brains were rapidly removed and placed in ice-cold isopentane solution, frozen and coronally dissected into 20 μm-thick slices on cryostat. Then, CMHs were counted blinded to treatment on each brain slice and characterized according to size as described previously (M. Pétrault et al., 2019). Briefly, CMHs that were evident to the eye aided by a 2x magnifying glass, and found in only 1 slice, were defined as type 1 CMHs. When evident to the naked eye and found again in the next slice, CMHs were defined as type 2. When larger and found again in more than 2 slices, CMHs were defined as type 3. A total hemorrhagic score was calculated as the sum of all CMHs found throughout the brain and considered as the CMH lesions extent. Mice with less than 10 CMHs were excluded from the study.

Statistical analysis

All values were expressed as mean ± standard error of the mean (SEM). Individual data points are superimposed on boxplots. For behavioral assessment, continuous variables were compared with a two-way ANOVA followed by a post-hoc Tukey's Multiple Comparison

Test if variance analysis was significant. For the CMH score, a Pearson's Chi-squared test was realized. In each case, a value of p<0.05 indicated statistical significance.

Results

Acute functional effect of CMHs in the brain of 14-month-old mice after a 12month diet

At 12 months of diet, mice on HFD endured metabolic disturbances with a significant increase in blood total cholesterol and visceral adipose tissue compared to ND-fed mice (Table A). Significant differences were found in the measured locomotor parameters only between HFD-fed mice with CMHs, compared with mice without CMHs fed with HFD or ND, including a decrease in the travelled distance (-39% vs ND mice, p=0.002; -31% vs HFD mice, p=0.02), an increased resting time (+92% vs ND mice, p=0.002; +68% vs HFD mice, p=0.005), and a decrease in the mean walking speed (-38% vs ND mice, p=0.004; +31% vs HFD mice, p=0.02, Fig. 1B). Concerning cognitive functions, the performances on spontaneous alternation test were not statistically different between all groups (p<0.05, Fig.

1C-left panel). On the NOR test, the onset of CMH in mice fed with the same diet did not change the performances significantly. A significant reduction was however found in HFDfed mice with CMHs compared with mice without CMHs and fed with ND (HFD + CMH -89% vs ND mice, p=0.014). A comparable drop in the performances was observed in HFDfed mice without CMHs compared with ND-fed mice with or without CMHs (HFD -106% vs ND mice, p=0.0015; HFD -107% vs ND + CMH mice, p=0.02; Fig. 1C-right panel).

Acute functional effect of CMHs in the brain of 5 month-old-mice after a 3month diet

At 3 months of diet, mice on HFD were metabolically disturbed, with a significant increase in weight, blood total cholesterol and visceral adipose tissue compared to ND-fed mice (Table A). Regarding spontaneous locomotor parameters, the travelled distance and the mean walking speed increased slightly but without reaching statistical significance in mice with CMHs compared with mice without CMHs (+17% and +18% respectively, p=0.06 for both,

Acute CMH lesions in the brain of 14 month-old-mice after a 12-month diet

After 12 months of diet, visual CMHs were found in all areas of the Sylvian territory of the brains of all mice injected with RAME-CD (Fig. 3A). In ND-fed mice, a mean number of 20 CMHs (19.57 +/-0.91) per brain was observed at 7 days after injection. Microhemorrhages were mostly of small size (type 1). Medium-sized CMHs (type 2) were counted to a maximum of 4. In HFD-fed mice, a mean number of 25 (25.08 +/-1.39) CMHs per brain was counted, mostly of small size (type 1). Medium sized CMHs (type 2) reached a maximum of 5 in two brains, and two larger CMHs (type 3) were observed in one of these brains. The number of CMHs induced in HFD-fed mice was significantly higher than in ND-fed mice (p=0.01), without a significant modification of the typology of CMHs (p=0.39, Fig. 3B).

Acute CMH lesions in the brain of 5 month-old-mice after a 3-month diet

After 3 months of diet, visual CMHs were found in all areas of the Sylvian territory of the brains of all mice injected with RAME-CD (Fig. 3C). One mouse was excluded of the study with only 9 CMH. In ND-fed and HFD-fed mice, mean numbers of 17 (17.40 +/-0.92) and 19 (19.00 +/-0.21) CMHs per brain were observed at 7 days after injection. Microhemorrhages were mostly of small size (type 1). The majority of ND-fed mice had between 1 and 2 medium-sized CMHs (type 2), and the majority of HFD-fed mice had between 2 and 3 CMHs of this size. The type 3-CMH was not observed. Neither the number (p=0.64) nor typology of CMHs (p=0.82) was significantly different between ND-and HFD-fed mice (Fig. 3D).

The comparison between the observed CMH lesions at 5 or 14 months of age (3 and 12 months of diet respectively) did not show any difference in ND-fed mice (p=0.92), whereas there was a significant increase in the number of CMHs in HFD-fed mice after 12 months of diet compared with HFD-fed mice after 3 months of diet (p=0.02, Fig. 3E-left panel).

However, no significant difference was observed in the typology of CMHs, whatever the group tested (p=0.90 and 0.53 respectively, Fig. 3E-right panel).

Long-term functional effect of CMHs

Throughout the year following the onset of CMHs, the spontaneous locomotor parameters of mice (Fig. 4B) did not change significantly in regard to the travelled distance, the resting time or the mean walking speed. Regarding cognitive functions, the performances on the spontaneous alternance test did not change with time for all groups of mice (Fig. 4C

Progression of the CMH lesions

The mortality observed during the 12-month period did not differ significantly among groups with CMHs, and no hematoma was found in the brain of prematurely died animals, even if the microhemorrhagic score obtained in some of dead mice was high (Fig. S1). The CMHs were induced 3 months after the start of diet, when mice were 5 months old. The CMH count and profile of the survivors was determined 9 months after RAME-CD injection, when mice were eventually 14 months old. (Fig. 5A). In ND-fed and HFD-fed mice, mean numbers of 20 (19.57 +/-0.89) and 22 (22.00 +/-1.67) CMHs per brain were observed. Microhemorrhages were mostly of small size (type 1). One to three medium-sized CMHs (type 2) were found in the brains of ND-fed mice, and two to five in the brains of HFD-fed mice. One type 3 CMH was observed in each group. The number of visual CMH events increased slightly upon HFD, but was not significant (p=0.17). Likewise, HFD did not modify the typology of CMHs despite a slight increase in the proportion of type 2 CMHs (p=0.68, Fig. 5B). The comparison of the numbers of CMHs (Fig. 5C-left panel) and their typology (Fig. 5C-right panel) between onset (i.e. 7 days and 9 months after RAME-CD injection) did not show any significant difference (p>0.05).

Discussion

The main finding of our study discloses that induced CMHs lesions have differential impact on locomotor and cognitive functions with age and diet, and do not precipitate cognitive decline at midlife.

We first aimed at determining the acute effects of CMHs on midlife (14-month-year old) mice after a 12-month ND or HFD, because the prevalence of the associated metabolic disturbances remains high in middle-aged humans, around 35% of the general population [START_REF] Aguilar | Prevalence of the Metabolic Syndrome in the United States, 2003-2012[END_REF][START_REF] Bhosale | A case control study of clinical and biochemical parameters of metabolic syndrome with special attention among young and middle aged population[END_REF]. In our model, a 40% HFD induced a progressive increase in weight, blood lipid profile and visceral adipose tissue (Table A). Despite being incompletely explored, these disturbances can be considered metabolic in nature, becoming significant from 3 months of diet as already reported (O. Pétrault et al., 2019). We define the severity of the induced microhemorrhagic lesions both by the total number of CMHs, reflecting the number of microvascular events, and by the profile (typology) reflecting the extend of blood effusion (M. Pétrault et al., 2019). The increase observed in the number of induced CMHs in midlife mice on HFD compared with mice of the same age on ND suggests the establishment of an enhanced microvascular sensitivity to a mechanical erosion process with HFD. Indeed in our method, RAME-CD induce bleeding through the erosion of the endothelial luminal membrane (M. Pétrault et al., 2019). A greater endothelial sensitivity to blood shear stress can be hypothesized since reported upon various cardiovascular risk factors, although this has to be clarified at the brain microvessels [START_REF] Borshchev | Pathogenetic pathways of cognitive dysfunction and dementia in metabolic syndrome[END_REF][START_REF] Granger | Microvascular responses to cardiovascular risk factors[END_REF][START_REF] Lubrano | Roles of LOX-1 in microvascular dysfunction[END_REF]. This microvascular fragility does not seem to be of critical extent as in structurally diseased vessels, because neither spontaneous lesion nor remodeled vascular wall has been observed in our mice, supporting by our histology investigations (Fig. S2). We rather consider a disturbance of the vascular endothelial cell metabolism and function, as found in the literature and in our previous study [START_REF] Li | Early effects of high-fat diet on neurovascular function and focal ischemic brain injury[END_REF][START_REF] Medeiros | Arginine and aerobic training prevent endothelial and metabolic alterations in rats at high risk for the development of the metabolic syndrome[END_REF]O. Pétrault et al., 2019). This change in the lesions when mice are on HFD was accompanied by a potentiated impairment of the spontaneous locomotion, found not significant in mice on ND.

This impairment was reflected by a decreased travelled distance and mean walking speed, associated with an increased resting time. Therefore, in our conditions, middle age alone was not enough to render CMHs symptomatic. Only one year of metabolic disturbances seem to facilitate the emergence of acute locomotor symptoms when CMHs occur. Of course, whether such symptoms would be of enough magnitude to lead to a neurologic complaint in humans is far from evidenced. Conversely, the induction of CMHs neither altered the working memory nor the visual recognition memory in both diet conditions. The impairment of the visual recognition memory already induced by one year of HFD was not aggravated either. Whether CMHs induced at midlife could have consequences on mice along the second half of their life span needs further and quite delicate investigations. Other cognitive disturbances could be detected using more sensitive behavioral tests, as Barnes maze. In spite of the reported visual recognition memory impairment in patients in the early course of mild cognitive impairment [START_REF] Barbeau | Evaluation of visual recognition memory in MCI patients[END_REF], an extrapolation to humans needs caution and further studies.

The onset of CMHs in young adult (5-month-old) mice after 3 months of HFD, induced similar microhemorrhagic lesions to that of the ND mice, but remained less numerous than the ones observed in HFD mice experiencing acute CMHs at midlife. This confirms that a long time of metabolic disturbances may represent a higher risk of extended CMH number. The CMHs seem to have a slight stimulant effect, in both groups, reflected by a significant decrease in the resting time. This result is consistent with another study carried out in female mice under HFD in which a temporary increase in the locomotor activity was observed and interpreted as an increased anxiety [START_REF] Krishna | Time-dependent behavioral, neurochemical, and metabolic dysregulation in female C57BL/6 mice caused by chronic high-fat diet intake[END_REF]. This trouble caused by CMHs was concomitant with an altered visual recognition memory. At this diet duration, we had previously demonstrated that HFD reflects the start of metabolic disturbances and does not alter the brain functions tested here (O. Pétrault et al., 2019). This enabled us in the present study to observe the proper detrimental action of CMHs on cognition. Surprisingly, the acute effect of CMHs on cognition was independent on the diet and even statistically significant only in the ND condition. Since our previous work on younger mice (2 months) revealed completely silent CMHs at onset (M. Pétrault et al., 2019), the age of the present mice (5months-old) might have been a more potent risk factor of CMHs symptomatology in the acute phase than the start of the metabolic disturbances. This is also surprising since CMHs exerted no effect in our 14-month-old mice on ND. An earlier life period can be more vulnerable to deleterious stimuli affecting mnesic functions, as reported for HFD itself in some experimental situations [START_REF] Boitard | Juvenile, but not adult exposure to high-fat diet impairs relational memory and hippocampal neurogenesis in mice[END_REF]. These acute symptoms might also be explained by our method which induces a consistent average pattern of 20 CMHs in seven days. We thus investigated whether these acute effects are transient or evolving in time.

Recently, CMHs have shown long-term effects on cognition [START_REF] Bergeron | Role of cortical microbleeds in cognitive impairment: In vivo behavioral and imaging characterization of a novel murine model[END_REF]M. Pétrault et al., 2019). In our study, 5-month-old mice that had been fed for 3 months with ND or HFD, were induced CMHs and kept for a prolonged diet during 9 months after CMHs onset, so up to midlife (14 months of age). A prolonged HFD did not significantly influence the development of new CMHs possibly resulting from a rebleeding of previous CMHs or new onset CMHs, although the apparition of a type 3 CMHs was observed in each group at 14 months of age compared with the acute CMHs profiles at 5 months of age. However, the comparisons between CMH scores must be considered with caution since from different mice groups. Such a stability observed in the bleedings overtime differs from the large intracerebral hemorrhage situation, where the presence of metabolic disturbances increases the risk of onset and severity [START_REF] Guo | Hyperglycemia and Mortality Risk in Patients with Primary Intracerebral Hemorrhage: A Meta-Analysis[END_REF][START_REF] Park | The relationship between metabolic syndrome (MetS) and spontaneous intracerebral hemorrhage (ICH)[END_REF], in link with elevated blood pressure, also reported in a microbleeding context recently [START_REF] Mitaki | Metabolic syndrome is associated with incidence of deep cerebral microbleeds[END_REF][START_REF] Toth | Aging exacerbates hypertensioninduced cerebral microhemorrhages in mice: role of resveratrol treatment in vasoprotection[END_REF]. Although blood pressure was not measured here, we may speculate that our HFD conditions authenticating imbalanced lipid metabolism (Table A) did not have a sufficient effect on microvessel wall remodeling or on local hemostasis to aggravate the CMHs in number or extent. Hence, metabolic disturbances are rather considered as prothrombotic, in particular since hyperplasia and hypertrophy of adipocytes may lead to the overexpression of adipocytokines and plasmatic PAI-1 [START_REF] Mccracken | Pathophysiology of the metabolic syndrome[END_REF], thus limiting the activity of tissue-type plasminogen activator (tPA) and the extension of the bleeding. The alteration of the spontaneous locomotion induced by CMHs at 5 months of age was not found further, confirming its transient nature. The working memory was not impaired. The visual recognition memory was significantly impacted by the presence of CMHs or HFD only from 11 months of age (9 months of diet) as previously described (M. Pétrault et al., 2019;O. Pétrault et al., 2019). Mice combining HFD and CMHs were functionally impaired with the same kinetic, suggesting no synergistic effect, therefore the same underlying pathophysiological mechanism. Otherwise, one might have expected a deficit in the visual recognition memory at earlier time points, i.e. 8 or 11 months of age. As our mice did not show a progression of CMH lesions towards larger hemorrhages, the onset of such a 20-CMH burden in average does not seem to behave like a stroke which is a major cause of dementia.

Nevertheless, whether the precipitation of cognitive decline relies on the increase in the microlesions number or extent is still under question. Yet in humans, only the accumulation of CMHs above 2% per year [START_REF] Poels | Incidence of Cerebral Microbleeds in the General Population: The Rotterdam Scan Study[END_REF] evokes a potential progression towards cognitive decline, underlying a cerebrovascular or neurodegenerative disease. The potential mechanisms leading to functional impairment may include an alteration of the neurovascular coupling, targeting either the vascular endothelial cells or the smooth muscle cells, leading to an interruption in the retrograde propagation of neurovascular coupling [START_REF] Iadecola | The Neurovascular Unit Coming of Age: A Journey through Neurovascular Coupling in Health and Disease[END_REF]. As our method and conditions target the endothelium, CMHs or metabolic disorders could therefore interfere with this spread and thus limit vascular responses which could impact the cognitive performance in the long term [START_REF] Iadecola | The Neurovascular Unit Coming of Age: A Journey through Neurovascular Coupling in Health and Disease[END_REF][START_REF] Mogi | Neurovascular Coupling in Cognitive Impairment Associated With Diabetes Mellitus[END_REF].

Neuroinflammation can also be implied in both stresses, as CMHs [START_REF] Ahn | Diverse Inflammatory Response After Cerebral Microbleeds Includes Coordinated Microglial Migration and Proliferation[END_REF] and metabolic disorders are known to induce inflammation via the activation of astrocytes and microglia and the release of cytokines [START_REF] Van Dijk | Integrative neurobiology of metabolic diseases, neuroinflammation, and neurodegeneration[END_REF][START_REF] Van Dyken | Impact of Metabolic Syndrome on Neuroinflammation and the Blood-Brain Barrier[END_REF].

More work including a diet or pharmacological modulation might help determine the mechanisms involved. But before that, technically challenging investigations on the very long-term consequences of early or midlife CMHs onset are needed to define their role on late-life dementia in link with metabolic disturbances. Indeed, very long-term experiments including mice with lesioned brains often result in a high spontaneous mortality rate [START_REF] Selim | (HEADS) Roundtable Participants[END_REF].

Taken together, our results suggest that contrary to hemorrhagic stroke, the onset of CMHs in the young adult stage can be slightly and transiently symptomatic but neither evolves towards larger lesions nor a precipitated impairment of the visual recognition memory after a prolonged HFD up to midlife. However, the onset of CMHs at midlife induces significant locomotor symptoms only under HFD. Research on the consequences of CMHs should consider that even within the midlife range, age plus duration of diet-induced metabolic disturbances enhance the outcome of CMHs.

Acknowledgements

We warmly thank Prof Anne Muhr-Tailleux for her great technical assistance in blood assays

Fig

  Fig.2B). The resting time was yet significantly reduced by CMH onset in both ND-(-53% vs HFD mice, p=0.03) and HFD-fed mice (-50% vs HFD, p=0.03). Regarding cognitive functions, the performances on the spontaneous alternance test did not change among groups (p<0.05, Fig.2C-left panel). However, a significant reduction in the performances on the NOR test was observed in mice with CMHs and fed with ND (-44% vs ND mice, p=0.006) or HFD (-40% vs ND mice, p=0.015) compared with mice without CMHs and fed with ND (Fig.2C-right panel).

  , left panel). The performances on the NOR test of ND-fed mice without CMHs was preserved during the whole protocol, with a stable discrimination NOR index (Fig.4C, right panel). The induction of CMHs in ND-fed mice reduced these performances from 11 months of age until significance at 14 months of age (-69% vs ND mice, p=0.05). The NOR index of HFD-fed mice decreased progressively until 14 months of age (12 months of diet) and became significantly different from ND-fed mice from 11 months of age (9 months of diet, -80% vs ND mice, p=0.03). The presence of CMHs in HFD-mice did not precipitate the onset of the decrease of NOR index compared with HFD-mice without CMH. The deficit of visual recognition memory is similar in both HFD groups with a score index significantly reduced in comparison to ND-fed mice without CMHs (-113 % vs ND mice, p=0.001).

(Figure 1 :

 1 Figure captions

Figure 2 :

 2 Figure 2: Acute effects of cerebral microhemorrhages on locomotor and cognitive functions of 5 month-old mouse (3 months of diet). A. Short-term test protocol for the acute effects of the CMH lesions induced at 5 month-old (after 3 months of diet, n=7-8 per group). B. Spontaneous locomotor behavior of mice. No significant differences were observed in the travelled distance (left, in cm) and mean walking speed (on the right, in cm/s) between groups. A significant decrease in the resting time (in second) was observed among mice with CMHs (ND+CMH and HFD+CMH). C. Working memory (spontaneous alternation index) and visual recognition memory (novel object recognition index) were examined through the spontaneous alternation test and the novel object recognition (NOR) test respectively. No difference was found in the performance on the spontaneous alternation test between groups. A significant difference was found in the performance on the NOR test in mice with CMHs (ND+CMHs and HFD+CMHs) compared to ND mice. Date are shown as mean ± sem, individual data points are superimposed on boxplots. Statistical analysis was performed using a two-way ANOVA followed by a Tukey's post-hoc test. *p < 0.05, **p<0.01. ND: Normal Diet. ND+CMH: Normal Diet with CMHs. HFD: High-Fat Diet. HFD+CMH: High-Fat Diet with CMHs.

Figure 3 :

 3 Figure 3: Acute microbleedings of 14 month-old mice (12 months of diet) and 5 month-old mice (3 months of diet). A. Representative view of cerebral microhemorrhages (CMHs) in

Figure 4 :

 4 Figure 4: Long-term effects of cerebral microhemorrhages on mouse locomotor and cognitive functions. A. Long-term test protocol for the follow-up of the effects of early CMHs in the brain of mice after 12 months of diet (n=10-12 per group). B. Spontaneous locomotor behavior, reflected by the travelled distance (in cm), the resting time (in second) and the mean walking speed (in cm/s). No significant difference was observed between groups. C. Working memory (spontaneous alternation index) and visual recognition memory (novel object recognition index) were examined through the spontaneous alternation test and the novel object recognition (NOR) test respectively. No difference was found in the performance on spontaneous alternation test between groups. Several mice were excluded of the test according to the exclusion criteria (n=1-2 per group and time). Visual recognition memory of ND-fed mice was preserved during the protocol, as given by the stable discrimination NOR index. The induction of CMHs in ND-fed mice reduced these performances until significance from 14 months of age (12 months of diet). The NOR index of HFD-fed mice decreased progressively until 14 months of age and became significantly different from ND-fed mice after 11 months of age (9 months of diet). The presence of CMHs in HFD-fed mice resulted in a significantly reduced NOR index at 11 months of age compared with ND control group. Several mice were excluded of the test according to the exclusion criteria (n=1-2 per group and time). Date are shown as mean ± sem, individual data points are superimposed on boxplots. Statistical analysis was performed using a two-way ANOVA followed by a Tukey's post-hoc test. *p < 0.05, **p<0.01, ***p<0.001. ND: Normal Diet. ND+CMH: Normal Diet with CMHs. HFD: High-Fat Diet. HFD+CMH: High-Fat Diet with CMHs.

Figure 5 :

 5 Figure 5: Cerebral microhemorrhage profiles of 14 months-old mice on prolonged diet, 9 months after onset. A. Representative view of cerebral microhemorrhages (CMHs) in 14

  Mosaic plot of CMHs in each group. These CMH profiles were not different between both groups according to Pearson's Chi-squared test (p=0.82). E. Time comparison of acute microbleedings at 5 month-old (3 months of diet) or 14 month-old (12 months of diet). The mean numbers of CMHs were not significantly different between both ND-fed mice groups (p=0.92), but significantly increased in HFD-fed mice at 14 months of age (after 12

	months of diet) compared with 5 months of age (after 3 months of diet) according to two-way
	ANOVA followed by a Tukey's post-hoc test (p=0.02). The CMH profiles were not different
	among ND-fed mice (p=0.90) and HFD-fed mice (p=0.53) according to the Pearson's Chi-
	squared test. Date are shown as mean ± sem, individual data points are superimposed on
	boxplots. ND: Normal Diet. HFD: High-Fat Diet.
	mice after 12 months of normal diet (ND) or high-fat diet (HFD), and the dot plots of CMHs
	for each mouse (n=12-15 per group). B. Mean number of CMHs at onset after 12 months of
	diet. A significant increase was found in HFD-fed mice compared with ND-fed mice (Student
	test, p=0.01). Mosaic plot of CMHs in each group. The ratio refers to the percentage of each
	type of microhemorrhage among all the observed CMHs. These CMH profiles were not
	different between both groups according to Pearson's Chi-squared test (p=0.39). C.
	Representative view of CMHs in mice at 5 months of age, after 3 months of ND or HFD, and

). A. Representative view of cerebral microhemorrhages (CMHs) in the dot plots of the CMHs for each mouse (n=6-7 per group). D. Mean number of CMHs at onset after 3 months of diet. No significant difference was obtained between groups (Student test, p=0.64).


Fengming Qiang

Emmanuel Bouzy
email: emmanuel.bouzy@univ-lorraine.fr

Hongchao Kou
email: hchkou@nwpu.edu.cn

Yudong Zhang

Lingling Wang

Jinshan Li

Grain fragmentation associated continuous dynamic recrystallization (CDRX) of hexagonal structure during uniaxial isothermal compression: High-temperature α phase in TiAl alloys

Keywords: TiAl alloys, Continuous dynamic recrystallization (CDRX), Grain boundary bulging, Grain fragmentation, Intragranular Misorientation Axis (IGMA) analysis

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

Introduction

Thermomechanical processing is an important technique for metal forming. During the process, crystal deformation and restoration, i.e., recovery and recrystallization, interweave dynamically, resulting in specific microstructural evolution and mechanical property change.

Thus, the restoration mechanisms of "dynamic recovery (DRV)" and ''dynamic recrystallization (DRX)" [1][START_REF] Huang | A review of dynamic recrystallization phenomena in metallic materials[END_REF][START_REF] Sakai | Dislocation substructures developed during dynamic recrystallisation in polycrystalline nickel[END_REF][START_REF] Cram | Modelling discontinuous dynamic recrystallization using a physically based model for nucleation[END_REF][START_REF] Sakai | Plastic Deformation: Role of Recovery and Recrystallization[END_REF][START_REF] Doherty | Current issues in recrystallization: A review[END_REF][START_REF] Sakai | Dynamic and post-dynamic recrystallization under hot, cold and severe plastic deformation conditions[END_REF] have been a subject of intensive study for decades.

Previously, most of the investigations were conducted on the DRX behavior of cubic materials. Discontinuous dynamic recrystallization (DDRX) during which new DRXed grains nucleate and grow at the expense of the work-hardened surroundings has been found to be dominant in FCC materials with low or medium stacking fault energy [START_REF] Wusatowska-Sarnek | Nucleation and microtexture development under dynamic recrystallization of copper[END_REF][START_REF] Belyakov | Dynamic recrystallization under warm deformation of a 304 type austenitic stainless steel[END_REF][START_REF] Brünger | Nucleation mechanisms of dynamic recrystallization in austenitic steel alloy 800H[END_REF][START_REF] Dehghan-Manshadi | Recrystallization in AISI 304 austenitic stainless steel during and after hot deformation[END_REF][START_REF] Ponge | Necklace formation during dynamic recrystallization: Mechanisms and impact on flow behavior[END_REF]. During the deformation process, bulging of grain boundaries was frequently observed and regarded as a prelude for nucleation. Further analysis revealed that the bulging was operated by a mechanism closely related to the strain-induced grain boundary migration. In such a case, a significant dislocation density difference existed between the front and the behind of the bulged boundary and the migration direction was from the low density region to the high density region [1,[START_REF] Roberts | A nucleation criterion for dynamic recrystallization during hot working[END_REF]. Then, a necklace of new grains formed along the boundary [START_REF] Belyakov | Dynamic recrystallization under warm deformation of a 304 type austenitic stainless steel[END_REF][START_REF] Brünger | Nucleation mechanisms of dynamic recrystallization in austenitic steel alloy 800H[END_REF][START_REF] Dehghan-Manshadi | Recrystallization in AISI 304 austenitic stainless steel during and after hot deformation[END_REF][START_REF] Ponge | Necklace formation during dynamic recrystallization: Mechanisms and impact on flow behavior[END_REF].

However, for BCC metals and FCC metals with high stacking fault energy, continuous dynamic recrystallization (CDRX) was frequently observed [START_REF] Gourdet | A model of continuous dynamic recrystallization[END_REF][START_REF] Sakai | Continuous dynamic recrystallization during the transient severe deformation of aluminum alloy 7475[END_REF]. The low angle grain boundaries were formed during deformation by DRV and evolve into high angle grain boundaries through progressive accumulation of dislocations. Necklace microstructure was also produced by CDRX at an intermediate stage owing to the rapid development of strain gradients near grain boundaries [START_REF] Sakai | Continuous dynamic recrystallization during the transient severe deformation of aluminum alloy 7475[END_REF][START_REF] Musin | Microstructure evolution in a cast 1421AI alloy during hot equal-channel angular extrusion[END_REF].

For the hexagonal structured materials, such as the Mg alloys, commercially pure Ti (CP-Ti) and TiAl-based alloys (high-temperature α phase) [17], that have drawn considerable industrial attention owing to their light-weight. Due to the limited available slip systems, they should possess specific microstructural characteristics when DRX takes place. The available investigations mainly focused on Mg alloys and CP-Ti. It was found that Mg alloys usually display typical features of CDRX during hot deformation [START_REF] Yang | Effect of prior strain on continuous recrystallization in AZ31 magnesium alloy after hot deformation[END_REF][START_REF] Yang | Annealing behavior of a cast Mg-Gd-Y-Zr alloy with necklace fine grains developed under hot deformation[END_REF][START_REF] Yang | Dynamic recrystallization and texture development during hot deformation of magnesium alloy AZ31[END_REF][START_REF] Yang | Dynamic nucleation of new grains in magnesium alloy during hot deformation[END_REF][START_REF] Miura | Mechanisms of grain refinement in Mg-6Al-1Zn alloy during hot deformation[END_REF], i.e., low angle boundaries were introduced due to the accumulation of dislocations, and then new fine grains were developed progressively from grain boundaries to grain interiors with the gradual increase in misorientation at sub-boundaries. Due to the lack of slip systems, twinning [START_REF] Miura | Mechanisms of grain refinement in Mg-6Al-1Zn alloy during hot deformation[END_REF][START_REF] Sitdikov | Dynamic recrystallization in pure magnesium[END_REF][START_REF] Barnett | Influence of grain size on the compressive deformation of wrought Mg-3Al-1Zn[END_REF][START_REF] Galiyev | Correlation of plastic deformation and dynamic recrystallization in magnesium alloy ZK60[END_REF][START_REF] Miura | High temperature deformation and extended plasticity in Mg single crystals[END_REF] and kinking [START_REF] Yang | Annealing behavior of a cast Mg-Gd-Y-Zr alloy with necklace fine grains developed under hot deformation[END_REF][START_REF] Yang | Dynamic recrystallization and texture development during hot deformation of magnesium alloy AZ31[END_REF][START_REF] Yang | Dynamic nucleation of new grains in magnesium alloy during hot deformation[END_REF][START_REF] Miura | Mechanisms of grain refinement in Mg-6Al-1Zn alloy during hot deformation[END_REF] served as additional mechanisms for rapidly introducing large misorientations in deformation substructures and accelerating the formation of new fine grains. For the DRX behavior of CP-Ti, investigations [START_REF] Chao | Deformation behaviour of a commercial pure titanium alloy during hot compression testing[END_REF][START_REF] Zeng | Deformation behaviour of commercially pure titanium during simple hot compression[END_REF][START_REF] Yan | Hot deformation and grain refinement mechanisms of commercially pure titanium processed via three-directional cryo-compression[END_REF] indicated that DRV was the dominant softening process for the annihilation of dislocation during high temperature deformation. The original grain boundaries become serrated with the simultaneous formation of internal subgrains. With further deformation, the microstructure was continuously recrystallized resulting in a fine DRX grain microstructure.

For TiAl alloys, due to the specific Blackburn orientation relationship [START_REF] James | The Science, Technology and Application of Titanium[END_REF] (Blackburn OR: 111 // 0001 , 〈11 0〉 //〈112 0〉 ), the features of the lamellar structure (α2+γ), such as colony size, lamellar orientation, directly depend on the high-temperature α phase. Therefore, it is necessary to investigate the DRX behavior and its induced microstructure evolution of the high-temperature α phase during thermomechanical processing. Till now, only several researches [START_REF] Schwaighofer | Hot-working behavior of an advanced intermetallic multi-phase γ-TiAl based alloy[END_REF][START_REF] Schmoelzer | An in-situ highenergy X-ray diffraction study on the hot-deformation behavior of a β-phase containing TiAl alloy[END_REF][START_REF] Liss | In situ study of dynamic recrystallization and hot deformation behavior of a multiphase titanium aluminide alloy[END_REF] based on in-situ high-energy X-ray diffraction (HEXRD) characterization technique have been conducted. It has been revealed that the α phase was deformed preferentially by slip than by twinning, and the deformation was superimposed by a relatively slow DRV and a less pronounced DRX process. However, no microstructural evolution information was obtained, and the restoration behavior needs to be further investigated. For the high-temperature α phase in TiAl-based alloys, the situation is different from Mg alloys and CP-Ti, and the investigation is much difficult. On one hand, it is impossible to visualize the microstructures in the required temperature range by in-situ microscopy. On the other hand, because of the narrow single α phase region, the microstructure produced by hot deformation cannot be preserved completely to room temperature even if rapid cooling is performed [START_REF] Dey | Crystallography and phase transformation mechanisms in TiAl-based alloys -A synthesis[END_REF]. It undergoes a phase transformation from α phase to lamellar structure (α2+γ) during cooling [17].The dislocation structures produced during hot deformation are destroyed by the formation of the γ lamellae inside the α grains.

Fortunately, the α → α2+γ phase transformation does not destroy the α grain boundaries and the intragranular low-angle boundaries formed during hot deformation. Therefore, the high temperature deformed microstructure can be reconstructed from the orientation data of the low temperature phase based on the specific phase transformation orientation relationship.

The detailed principles and methods have been reported by L. Germain et al. [START_REF] Germain | An advanced approach to reconstructing parent orientation maps in the case of approximate orientation relations: Application to steels[END_REF] and the techniques have become mature. To reveal the dislocation slip and the formation of the lowangle boundaries evolved from the produced dislocations, the intragranular misorientation axis (IGMA) analysis technique [START_REF] Chun | Distribution characteristics of in-grain Misorientation axes in cold-rolled commercially pure titanium and their correlation with active slip modes[END_REF][START_REF] Chun | Investigation of prism <a> slip in warm-rolled AZ31 alloy[END_REF][START_REF] Hadorn | Role of solute in the texture modification during hot deformation of Mg-rare earth alloys[END_REF][START_REF] Yamasaki | Crystallographic classification of kink bands in an extruded Mg-Zn-Y alloy using intragranular misorientation axis analysis[END_REF] has been proved powerful, especially for hexagonal materials with limited active slip systems, and has been successfully applied to analyze the dislocation type in CP-Ti [START_REF] Chun | Distribution characteristics of in-grain Misorientation axes in cold-rolled commercially pure titanium and their correlation with active slip modes[END_REF] and Mg alloys [START_REF] Chun | Investigation of prism <a> slip in warm-rolled AZ31 alloy[END_REF][START_REF] Hadorn | Role of solute in the texture modification during hot deformation of Mg-rare earth alloys[END_REF][START_REF] Yamasaki | Crystallographic classification of kink bands in an extruded Mg-Zn-Y alloy using intragranular misorientation axis analysis[END_REF]. The main assumption made in the IGMA analysis approach is that under the action of dislocation slip the crystal bends around a specific crystallographic axis (termed as ''Taylor axis'' [START_REF] Yamasaki | Crystallographic classification of kink bands in an extruded Mg-Zn-Y alloy using intragranular misorientation axis analysis[END_REF][START_REF] Maddin | Geometrical aspects of the plastic deformation of metal single crystals[END_REF][START_REF] Mironov | Microstructure evolution during warm working of Ti-6Al-4V with a colony-α microstructure[END_REF]). is parallel to × , in which is the dislocation Burgers vector and the slip plane normal. By this relation, the dominant slip mode in the deformed crystal can be ascertained simply by matching the Taylor axis for a given slip mode to the experimentally determined IGMA. So far, this method has been used in the cases where the deformation involved only one type of dislocation slip. For deformation that involves more types of dislocations or even boundary sliding, the method needs to be extended.

Based on such a situation, we conducted a thoroughly investigation on the DRV and DRX mechanisms of the high-temperature α phase in a TiAl alloy, Ti-44.81Al-3.96Nb-0.98Mo-0.15B (at. %). A simple uniaxial compression was applied to the alloy at 1280 °C where the alloy was composed of single α phase [START_REF] Schwaighofer | Microstructural design and mechanical properties of a cast and heat-treated intermetallic multi-phase γ-TiAl based alloy[END_REF]. The hot-compressed microstructures were analyzed based on the electron backscatter diffraction (EBSD) measurement data. The high-temperature α phase was reconstructed using the home-made software, Merengue 2 (as detailed in [START_REF] Germain | An advanced approach to reconstructing parent orientation maps in the case of approximate orientation relations: Application to steels[END_REF]), based on the measured EBSD data and the Blackburn OR. Furthermore, the IGMA method was extended to analyze the formation mechanisms of asymmetrical-tilt and tilt-twist low-angle boundaries. This study aimed at fully revealing the mechanisms of the DRV and the DRX of the hexagonal phase and developing a relevant analyzing methodology.

Experiments

The alloy used in this work is Ti-44.81Al-3.96Nb-0.98Mo-0.15B (at. %). It was fabricated by vacuum arc remelting (VAR) and cast into rods with dimensions of ∅12 mm × 180 mm, then hot isostatically pressed (HIP) at 1280 °C for 4 h under a pressure of 140 MPa in argon atmosphere. Cylindrical specimens with dimensions of ∅10 mm × 15 mm were cut out of the HIPed cast rods by electric-discharge machining for uniaxial compression experiments. The uniaxial compression experiments were carried out in a Gleeble 3500 simulator under vacuum. The specimens were heated by the Joule effect under a lowfrequency current at a rate of 20 °C/s to 1280 ºC, and isothermally held for 10 min, then compressed at a strain rate of 0.01 s -1 to the respective true strains of 0.05, 0.07, 0.29 and 1.05.

The temperature was controlled by means of type S thermocouples spot-welded directly on the surfaces of the specimens. At the end of the compression, they were water-quenched to preserve the deformed microstructure. To obtain the initial microstructural information before the deformation, one specimen was held isothermally at 1280 ºC for 10 min in a resistance furnace and then water quenched.

The mesoscale microstructures were characterized using a JEOL 6500F scanning electron microscope (SEM) equipped with a field emission gun. The crystallographic features of the microstructures were characterized by EBSD using an Oxford EBSD camera and the Aztec online acquisition software package (Oxford Instruments). To achieve the surface quality for EBSD measurements, the specimens were first mechanically polished then electrolytically polished with a solution of 5% perchloric acid, 35% butan-l-ol and 60% of methanol at 35 V for 10 s at temperatures lower than 5 °C. The EBSD patterns were acquired at the accelerating voltage of 20 kV with a step size of 1 μm for coarse microstructure and a step size of 0.1 µm for fine microstructure. The γ phase was indexed using the tetragonal structure (P4/mmm) parameters. The online orientation determination was assisted with the pseudo-symmetry function [START_REF]Addressing pseudo-symmetric mis-indexing in the EBSD analysis of gamma-TiAl[END_REF] incorporated in the Aztec online acquisition software package to secure the reliability and accuracy.

In order to analyze the microstructure characteristics of the high-temperature α phase during compression, the transformed part of the α phase was reconstructed from the low temperature γ phase [START_REF] Germain | An advanced approach to reconstructing parent orientation maps in the case of approximate orientation relations: Application to steels[END_REF]. After reconstruction, the obtained orientation data of the α phase were analyzed with the Channel 5 (Oxford Instruments) to reveal the features of grain boundaries, substructures and intragranular disorientation. The α grain sizes were determined based on the reconstructed EBSD data, using the Channel 5 software. Firstly, the grain boundaries were identified under the criterion of that the disorientation angle between neighboring pixels is larger than 15°. Then, the diameter of a circle having equivalent area to that of the grain is defined as the grain size [START_REF]Grain size and grain boundary characterisation in SEM[END_REF]. To ensure a statistical relevance, the grain sizes and the area fractions of grains of interests were determined based on 2 to 4 EBSD maps each covering an area of 1.6 × 1.2 mm . Due to the unavoidable phase transformation from the high-temperature α to the γ lamellae that modified the local microstructural constituent, it is difficult to examine the dislocations in the deformed α phase by means of transmission electron microscopy (TEM). The corresponding dislocation activities were analyzed mainly by identifying the disorientation axis of low-angle boundaries and matching the axis with Taylor axes [START_REF] Yamasaki | Crystallographic classification of kink bands in an extruded Mg-Zn-Y alloy using intragranular misorientation axis analysis[END_REF][START_REF] Maddin | Geometrical aspects of the plastic deformation of metal single crystals[END_REF][START_REF] Mironov | Microstructure evolution during warm working of Ti-6Al-4V with a colony-α microstructure[END_REF] listed in Table A2-1 in Appendix 2.

Results and Discussion

3.1 Initial microstructure Fig. 1 (a1) shows the reconstructed as-thermally-treated α phase (1280 ºC/10 min/water quenching) that was the initial microstructure for the compression. For reference, the original EBSD band quality indexed micrograph demonstrating the γ lamellae formed during cooling within the α grains is displayed in Fig. 1 (a2). It can be seen from Fig. 1(a1) that the α grains are in equiaxed shape with an average grain size of 220 ± 84.0 μm. The disorientation angle distribution of the α phase in Fig. 1 (b) is in good agreement with the well-known Mackenzie distribution [START_REF] Mackenzie | Second paper on statistics associated with the random disorientation of cubes[END_REF] for materials with random crystal orientations, indicating that the initial α grains do not possess any preferred crystallographic orientations or texture. 3.2 Deformed microstructure 3.2.1 Grain boundary bulging and dynamic recovery (DRV) Fig. 2 shows the EBSD IPF micrographs of the specimen deformed to the true strain of 0.05 (reconstructed micrograph in Fig. 2 (a1) and original band quality indexed one in Fig. 2 (a2)) (The original non-reconstructed micrographs used in the figures hereafter will be given in Appendix 1). It is seen from Fig. 2 (a1) that although the α grains are still in equiaxed shape, remarkable changes happened to their boundaries. Serrations or bulges appeared along some α grain boundaries, as shown by the two examples outlined with black rectangular in Fig. 2

(a1).

One can infer that some parts of the grain sheared in one direction and extruded into the neighboring grain, and the other parts were subjected to an "extruding in" from the neighboring grain on the other side. Further local disorientation analysis represented by the Kernel average misorientation (KAM) micrograph displayed in Fig. 2 (b) demonstrates that numerous low-angle disorientations appear along the bulged boundary regions, suggesting that a large number of dislocations were accumulated in front of the bulges. Interestingly, in some locations, the densities of local disorientations inside the bulges are higher than those in front of the bulges, as indicated with the arrows (1 to 4) in Fig. 2 (b). This is opposite to the well-known strain-induced grain boundary migration, where grain boundaries migrate into high dislocation density regions, leaving the region behind with a lower dislocation density [1]. So, in the present work, the boundary bulging should be from a different origin. To find out the origin behind the boundary bulges, we further analyzed the orientation variation feature near the bulged boundaries. A representative example is shown in Fig. 3 (a).

The evolution of the disorientation with respect to the starting point along line L1 in Grain 1 (G1) from the grain interior (starting point) to the bulged parts is also displayed in the figure .   It is seen that the accumulated disorientation angle along L1 exhibits plateaus and jumps (1-3°). The disorientation angles and axes at A, B and C with respect to the orientation of the point before each jump are 1.8°/〈0001 〉, 1.2°/〈2 1 1 12 〉, and 2.4°/〈2 1 1 12 〉, respectively.

The maximum disorientation reaches 5.4°/〈0001 〉 when approaching the boundary.

Interestingly, the rotation axes are always close to 〈0001 〉. It is known that among the possible slip modes in the disordered hexagonal α phase [START_REF] Partridge | The crystallography and deformation modes of hexagonal close-packed metals[END_REF][START_REF] Britton | On the mechanistic basis of deformation at the microscale in hexagonal close-packed metals[END_REF], only the prismatic <a> provides a lattice rotation with the Taylor axis in the 〈0001 〉 direction [START_REF] Chun | Distribution characteristics of in-grain Misorientation axes in cold-rolled commercially pure titanium and their correlation with active slip modes[END_REF][START_REF] Chun | Investigation of prism <a> slip in warm-rolled AZ31 alloy[END_REF][START_REF] Hadorn | Role of solute in the texture modification during hot deformation of Mg-rare earth alloys[END_REF][START_REF] Mironov | Microstructure evolution during warm working of Ti-6Al-4V with a colony-α microstructure[END_REF] (see Table A2-1). This suggests that the deformation was mainly realized by the prismatic <a> slip. With this information, we further examine the relation between the bulge direction and the possible active slip system (slip plane and direction). Then, we plot the pole figures of the prismatic slip plane and the slip direction of G1 and display them in Fig. 3 (b). Among the three prismatic slip variants, the one possessing the highest Schmid Factor, -0.47, for compression is highlighted with the blue circles. This variant was in the favorable orientation to be activated during the deformation. The slip plane trace of this slip variant is also plotted with the black solid line in the 11 00 pole figure. It is found that the orientation of this trace is in reasonable coincidence with the bulging direction (Line L1 in Fig. 3 (a)). This suggests that the bulging of the boundary is related to the dislocation movement on this prismatic slip system.

Another microstructural phenomenon near the bulge regions is the formation of the segments of low-angle boundaries as shown by the abrupt change of the colors (marked with the lines at A, B and C) along L1 in Fig. 3 (a), where abrupt disorientation changes are present. This suggests that dislocation rearrangement happened accompanying the deformation. Further trace orientation analysis showed that those low-angle boundaries (lines at A, B and C) in Fig. 3 (a) are oriented very close to the trace of the 112 0 plane (the red line in the 〈112 0〉/ 112 0 pole figure in Fig. 3 (b)). This plane is just perpendicular to the active slip plane, i.e., the 11 00 plane. Such boundaries, in fact, align with the extra half planes of the prismatic <a> dislocations, as illustrated in Fig. 3 (c). Geometrically, a prismatic <a> edge dislocation is built by an extra half-plane of 112 0 that intersects the 11 00 prismatic slip plane on a line of 〈0001 〉 that is the dislocation line, as indicated in Fig. 3 (c).

For a low-angle boundary made up of the same type of edge prismatic <a> dislocations aligned normal to the prismatic slip plane, as the case in Fig. 3 (c), the rotation axis between the two parts separated by the boundary is coincident with the 〈0001 〉 direction and the boundary plane is marked by the extra half-planes of the dislocations, i.e., the 112 0 plane.

The situation in Fig. 3 (a) is typical and appears frequently in many other positions in the specimen. As the small deformation was performed at high temperature, 1280°C (0.85Tm), the atomic bonding is weakened and the vacancy concentration is increased. Thus, the disordered boundary regions could be further extended, i.e., the grain boundaries should be broadened, as illustrated in Fig. 4 (a). When the favorably oriented prismatic slip was activated in the α grains, dislocations of prismatic <a> were generated and moved through the grain interiors and arrived at grain boundary regions (Fig. 4 (b)). The broadened boundaries functioned as dislocation sinks with enhanced free volume. When one dislocation moved out of the grain and annihilated in the boundary, an atomic step of a is formed extruding to the boundary region. When large amount of the same typed dislocations on several bundles of parallel slip planes moved into the boundary region, the boundary becomes microscopically bulged (Fig. 4 (c)). Such a grain boundary bulging mechanism was also evidenced in a hot deformed AZ31 alloy [START_REF] Son | Mechanism of grain boundary serration during hot deformation of AZ31 alloy: Role of grain boundary dislocations and grain boundary sliding[END_REF].

With the deformation, more dislocations were multiplicated and started to accumulate in front of the grain boundaries, thus, under thermal agitation dislocation climb happened, initiating dislocation rearrangement process and then forming the low-angle boundaries in front of the bulged parts (Fig. 4 (d)). The fact that most of the low-angle boundaries at the present stage (true strain of 0.05) are characterized by a rotation around the 〈0001 〉 axis and the boundary plane coincides with the 112 0 plane demonstrates that only one prismatic <a> slip was mainly activated and the low-angle boundary is composed of the same type of edge dislocations, as illustrated in Fig. 4 (d). Such boundaries belong to the symmetrical-tilt boundary.

Formation of subgrain

When the true strain was increased to 0.07, almost all the grain boundaries were bulged.

Low-angle boundaries (3 -15°) frequently appeared near triple junctions or grain boundary bulges, as marked with the white lines in Fig. 5 (a). These low-angle boundaries tended to form a closed region with the bulged boundaries, suggesting that subgrains were forming along the bulged grain boundary regions. Detailed orientation analysis of the low-angle boundaries in terms of disorientation angle and the rotation axis in large sample areas (3 EBSD micrographs each covering an area of 1.6×1.2 mm 2 ) revealed that the disorientation axes of 92.6 % (in number) of low-angle boundaries fall into the following three categories, as also marked in Fig. 5 (a). The disorientation axis 〈0001〉 are the Taylor axis corresponding to the symmetrical-tilt boundaries formed by the arrangement of the prismatic <a> dislocations. These boundaries are mainly the ones that are not yet totally connected with the original grain boundary to form the subgrains, suggesting that in these regions the DRV is in progress. However, for the low angle boundaries with the disorientation axes of 〈101 〉 or 〈112 〉, they are usually connected with the bulging boundaries and the enclosed regions possess a much deviated orientation from that of the parent grain, becoming the subgrains separated from the parent grains. To analyze the formation process of the new kinds of boundaries, two representative examples were selected and are displayed in Fig. 5 (b) and(c). In the first case (Fig. 5 (b)), the disorientation between the subgrain and the original grain is 13.99°/〈1 011〉 , and in the second case (Fig. 5 (c)), the disorientation is 10.5°/〈112 1〉. Analysis revealed that in either case the grain bulging direction (marked with the green lines) is largely consistent with the trace of one prismatic slip plane, as highlighted with the green lines in the 11 00 pole figures in Fig. 5 (b) and(c). This indicates that the formation of the low-angle boundaries proceeded by two steps. The first was the formation of a tilt boundary with a disorientation of ' ( /) ( () ( = 〈0001〉) from single prismatic slip and dislocation rearrangement. The second was another rotation ' around ) of the bulged part.

The second rotation could be realized by the absorption of the dislocations of another slip system to the yet formed tilt boundaries ' ( /) ( . Thus, using the ' ( /) ( rotation (' ( = 1 -10°; ) ( = 〈0001〉) as the first rotation and the ' /) (' = 1 -10°; ) = corresponds to Taylor axis of one of the other slip systems in Table A2-1) as the second rotation, we tried to resolve the second one by calculating the total disorientation and then comparing it with the measured disorientation. The second rotation should be the one providing the minimum deviation of the calculated total disorientation from the measured disorientation. The corresponding Taylor axis ) reveals the newly activated slip system that contributed to the further evolution of the sub-grain boundaries. The detailed formulation to resolve the second rotation is given in Appendix 2. The calculation results show that for the ) = 〈101 〉 boundaries, the second rotation axis ) is 〈101 0〉, corresponding either to a basal <a> slip or a pyramidal II <c+a> slip (Table A2-1). More examples with disorientation axis of 〈101 〉 are displayed in Table A2-3 in Appendix 2. The occurrence of the basal slip is more probable because the CRSSs of prismatic, basal and pyramidal slip are ordered increasingly from the prismatic to the pyramidal slip [START_REF] Britton | On the mechanistic basis of deformation at the microscale in hexagonal close-packed metals[END_REF][START_REF] Bieler | The origins of heterogeneous deformation during primary hot working of Ti-6Al-4V[END_REF]. The basal slip has lower CRSS than the pyramidal slip, thus, under specific local stress, the basal slip could easily be activated. Further analysis show that the Schmid factors of the activated basal slip are not always very high. Even in some grains, all the absolute values of the Schmid factors of the basal slip is smaller than 0.3, indicating that the activation was mainly driven by local incompatible deformation between the neighboring grains in the boundary regions. These results confirmed that the appearance of the 〈101 〉 low-angle boundaries was indeed realized by two steps. The first is the formation of the symmetrical tilt low-angle boundaries with disorientation axis of <0001> by the rearrangement of the prismatic <a> dislocations, and the evolution of the symmetrical tilt boundaries to the asymmetrical low-angle boundaries by absorption of the basal <a> dislocations, as illustrated in Fig. 6 (a).

However, for the ) = 〈112 〉 boundaries, we tried the Taylor axis of all the possible slip systems in hexagonal structures, but could not find a reasonable match between the calculated total rotation and the measured disorientation. This means that the second rotation was not realized by dislocation activity but by a different mechanism. Thus, we further examined the orientation of the boundary plane. Interestingly, we found that although the disorientation axis of the boundaries was no longer 〈0001〉, the trace of the low-angle boundary (in white) is very close to the trace of the {11 2 0} plane, as indicated with the red line in the IPF micrograph and in the <112 0>/{112 0} pole figure in Fig. 5 (c). It means that the boundary plane still remained the {112 0} plane. As analyzed above (Fig. 3 (c)), this plane corresponded to the extra half-plane of the edge prismatic <a> dislocations formed during the first step, inferring that during the second step the evolution of the boundary could only be realized by a rotation around the axis normal to the low-angle boundary formed in the first step, i.e., <112 0> axis. Thus, using ) = 〈112 0〉 as the second rotation axis to calculate the total rotation, good matches appeared between the calculated dislocations and the measured ones, confirming that the second rotation was indeed realized by a rotation around the axis normal to the boundary.

More examples with disorientation axis of

〈112 〉 are displayed in Table A2-4 in Appendix 2.
This result evidenced that at the present stage of deformation grain boundary sliding happened through which the tilt symmetrical low-angle boundaries further evolved into the tilt-twist boundaries, as illustrated in Fig. 6 (b). As the sliding is rotational, we can denote such boundary sliding by rotational boundary sliding. By these two ways, the orientations of bulged parts continuously deviated from those of original grains, initiating the formation of the subgrains.

Grain growth

It should be mentioned that accompanying grain boundary bulging grain growth occurred during the deformation (up to ε=0.07). From the data given in Table 1, one can see that the average grain size increased from initially 220±84.0 μm (ε=0) to 326±160.2 μm when the specimen was deformed to ε=0.05, and further to 580±216.3 μm when the strain went up to ε=0.07. Interestingly, the values of the standard deviation were nearly doubled at ε=0.05

and almost tripled at ε=0.07. This indicates that the grain size distribution is very large. As at these two deformation stages, recrystallized grains did not form numerously, the wide grain size spread infers that grain growth was not uniform. This phenomenon is related to the early state of hot deformation. As at the two tiny strains, the imposed deformation was not unanimously dispatched to every grain. Some grains possessing favorably oriented slip systems were deformed, but the others without favorably oriented slip systems were less deformed or not deformed. For the former, boundary bulges were formed as a result of deformation. For the latter, the high compression temperature became the most important factor to promote grain boundary migration. Thus, certain grains grew by consuming the others, which resulted in the heterogeneous grain size distribution. 7 is the microstructure of the specimen deformed to the true strain of 0.29. It can be clearly seen that the microstructure exhibits bimodal grain structure, composed of the newly formed small grains and the remnants of the initial coarse grains (Fig. 7 (a)). The newly formed small grains with an average size of about 26 ± 11.4 µm (Table 1) are distributed along the initial grain boundaries, forming a necklace structure. When the strain increased from 0.07 to 0.29, the grain size of the retained coarse grains decreased, from an average size of 580 ± 216.3 µm to an average size of 490 ± 284.5 µm, and the area fraction of the small grains increased from 4.5% to 35.6% (Table 1). The KAM micrograph displayed in Fig. 7 (b) shows that the newly formed small grains indicated by the yellow arrows possess much less amount of local disorientations, thus, these small grains possess the feature of recrystallized grains. This suggests that the recrystallization process was realized by a continuous detachment of the subgrains from the grain boundary regions, and such a process occurred repeatedly toward grain interiors, demonstrating a CDRX feature. From the color features of the small grains along the coarse grain boundary regions in Fig. 7 (a), one can see that the small grains detached from the two neighboring coarse grains were further mixed during the deformation process. To reveal the mechanism of the mixing process of the recrystallized grains, the crystallographic orientation features of two examples of necklace areas, the one with a few small grains representing the beginning of detachment and the other with some more small grains representing the penetrating of the small grains into neighboring small grain regions displayed in Fig. 8, were further analyzed. It is seen from Fig. 8 (a) that the two coarse grains with distinct orientations (represented by the 0001 and 101 0 pole figures) were separated by a layer of small sized domains delimitated either by high-angle boundaries (black lines) or low-angle boundaries (white lines) or the mix of the two. This suggests that for these two coarse grains the formation of the "necklace" just started. Although the small domains were separated from the neighboring coarse grains by boundaries with disorientation angles higher than 15°, their orientations are characteristically either close to G1 or to G2. For example, in the EBSD IPF micrograph in Fig. 8 (a), the orientations of domains D1 and D2 (enclosed in the blue and black squares in the pole figures) are close to that of G1 and those of domains D3 and D4 (enclosed in the red and green circles in the pole figures) are close to that of G2.

Clearly these orientation domains were detached from the two coarse grains (G1 and G2) and mixed in the necklace layer. For the case shown in Fig. 8 (b), the thick "necklace" layer was formed around the coarse red grain. The 0001 and 11 00 pole figures in Fig. 8 (b) are from the small red domains and the coarse red grain in the EBSD Y axis IPF micrograph. One can see that the small red domains possess close crystallographic orientations to that of the coarse red grain. However, the small red domains were already separated by layers of small domains detached from the other neighboring coarse grains. This suggests that grain boundary sliding between the detached small domains happened, resulting in the mixing of these small grains. In this case the boundary sliding is both translational and rotational. The former allows the detached grains to spatially separate from their parent grain but does not affect their orientation. The latter is opposite. It does not make spatial separation but deviates their orientations from those of the parent. The evidence of the spatial separation of the new grains from the parent grain and their orientation deviation from the parent orientation infers the existence of the two types of boundary sliding. The occurrence of grain boundary sliding is quite reasonable under the present deformation conditions. Firstly, the deformation temperature (1280°C) was very high (0.8Tm), thus grain boundary strength was very low.

Secondly, the deformation strain rate was relatively low (0.01 s -1 ) that offered a sufficient time window for the slow process of sliding to occur, Thirdly, the sizes of the detached domains were relatively small that provided a high amount of boundary area. All these factors favored the occurrence of grain boundary sliding. This mechanism should also be an important deformation mechanism for the small grains to contribute to the external deformation and allow the spatial separation of the detached grains from their parent grains. It can be seen that the microstructure is mainly composed of nearly equiaxed small grains, indicating the completion of the recrystallization process. The average grain size is about 45 ± 23.9 μm. However, it is worth mentioning that in some relatively large grains there exist segments of high-angle boundaries connected with segments of low-angle boundaries, as marked with the black arrows in Fig. 9. This suggests that the grain fragmentation process was still in progress. The low-angle boundaries would further evolve into high-angle boundaries and eventually divided the coarse grains into differently oriented small grains. Although the nucleation of the recrystallized grains in the present work also started from subgrains as the recrystallized nuclei, it is quite different from the commonly reported nucleation modes related to subgrains, i.e., nucleation by sub-boundaries migration and nucleation by subgrain coalescence [START_REF] Rios | Nucleation and growth during recrystallization[END_REF]. There was neither subgrain boundary migration nor subgrain coalescence, but subgrain detachment from the parent grains by the increasing disorientation and grain boundary sliding. In consequence, the nuclei formed in this way were very large with sizes of several tens of micrometers and the recrystallization did not obviously undergo the grain growth stage. The recrystallization progressed (with the deformation) rather by continuous repetition of the formation of the subgrains and the detachment of the subgrains from grain boundary bulges, which developed from grain boundary region toward grain interior until all the initial coarse grains were fragmented. Such a feature should be related to the lack of slip systems of hexagonal crystals and also the slow deformation condition of the present work. During the deformation, only one or two slips systems were activated. On one hand, the produced dislocations could smoothly move to grain boundary regions without much hindrance, and on the other hand, no dislocation entanglement could occur in the grain interiors to form intragranular subgrains. As the subgrains is formed preferentially at the boundary regions, grain boundary sliding is much easier than in grain interiors. Thus, subgrain detachment (especially translational sliding) prevailed over sub-boundaries migration and subgrain coalescence.

Summary

The mechanisms of DRV and CDRX of high-temperature α phase in a Ti-44.81Al- During the first stage, the favorable prismatic slip systems were activated. Edge prismatic <a> dislocations were generated and piled up near grain boundaries. Under the unidirectional compression, grain boundaries that were not normal to the compression axis were broadened due to the tension in the direction perpendicular to the compression axis.

Dislocations were annihilated in the broadened boundary and formed steps bulging into the neighboring grains, resulting in bulging boundaries. Due to thermal agitation, accumulated dislocations also rearranged by dislocation climb accompanying the boundary bulging.

Symmetrical-tilt boundaries characterized by a disorientation axis of <0001> were formed. The results of the present work provided original information of the CDRX of hexagonal structured materials. The extended IGMA analysis method developed in the present study is applicable to the related investigations on deformation behavior of high-temperature phases when dislocations could not be preserved to room temperature. In the two cases, the first rotation is ' ( /) ( () ( = 〈0001〉 and we assume that ' ( = 1 to 10°. Supposing that the second rotation ) corresponds to a Taylor axis of another slip system (as listed in Table A2-1) or to the axis normal to the extra half-plane of the corresponding boundary dislocations (as listed in Table A2-2), and ' = 1 to 10°. The total rotation G ('/) can be calculated. Then the calculated G is further compared with the measured G ex ('/) ex , the ' /) provides the smallest deviation (∆') from the experimental G ex ('/) ex is assessed as the second rotation. We further simulated the combination of the two rotations ' ( /) ( ' ( = 1 -10°; ) ( = 〈0001〉 and ' /) ' = 1 -10°; ) = 〈101 0〉 EF 〈112 0〉 that produces a total rotation '/). The results are shown with the variation of ' as a function of ' ( and ' and with the stereographic projection of ) as a function of the ' ( /' ratio in Fig. A2-2. When ) = 〈101 0〉, it is seen that the two rotation angles ' ( and ' have a symmetrical contribution with respect to ' ( /' = 1 to the total disorientation angle. For the rotation axis, whatever the two rotations The above results demonstrate that the extended IGMA approach is capable of revealing the mechanisms driving the evolution of the low-angle boundaries, when direct dislocation examination is not possible for the material.

Fig. 1

 1 Fig. 1 Initial microstructure. (a1) EBSD Y axis inverse pole figure (Y-IPF) micrograph of the reconstructed high-temperature α phase; (a2) EBSD band quality indexed micrograph before reconstruction; (b) disorientation angle distribution of α phase.

Fig. 2

 2 Fig. 2 Deformed high-temperature α microstructure at true strain of 0.05. (a1) EBSD Y axis inverse pole figure (IPF) micrograph of α phase; (a2) original EBSD micrograph with band quality indexed contrast. The

Fig. 3

 3 Fig. 3 (a) EBSD Y axis IPF micrograph of a representative example of boundary bulge regions and the correlated disorientations (in angle and axis) along L1 in grain G1. (b) Prismatic slip plane and direction

Fig. 4

 4 Fig. 4 Schema illustrating the formation of boundary bulging by dislocation slip and the formation of symmetric-tilt low-angle boundary by dynamic recovery (DRV). (a) Grain boundary broadening; (b) activation of dislocation slip and dislocation pile-ups in front of broadened boundary; (c) annihilation of dislocations at grain boundary and formation of boundary bulge; (d) rearrangement of dislocations to form symmetrical-tilt low-angle boundary.

  (ⅰ) 〈0001〉 (39.4%), marked in white in the figure; (ⅱ) 〈101 〉 | | ≥ 0) (32%), marked in black in the figure; (ⅲ) 〈112 〉 | | ≥ 0 (21.2%), marked in yellow in the figure.

Fig. 5

 5 Fig. 5 Microstructural and crystallographic features of α grains in specimen deformed to true strain of 0.07. (a) EBSD Y axis IPF micrograph of high-temperature α phase; (b) EBSD Y axis IPF micrograph of the area outlined with blue rectangle in (a), and pole figures of prismatic slip plane and direction ( 11 00 and 〈112 0〉) and the disorientation axis 〈1 011〉 in G1; (c) EBSD Y axis IPF micrograph of the area outlined with black rectangle in (a), and pole figures of prismatic slip plane and direction ( 11 00 and 〈112 0〉) and the disorientation axis 〈112 1〉 in G2.

Fig. 6

 6 Fig. 6 Schema illustrating the evolution of low-angle boundaries with further deformation. (a) Asymmetrical-tilt boundaries with disorientation of '/) ) = 〈101 〉) formed by the first rotation ' ( /) ( () ( = 〈0001 〉) and the second rotation ' /) () = 〈11 00〉); (b) tilt-twist boundary with the disorientation of θ/T () = 〈112 〉) formed by the first rotation ' ( /) ( () ( = 〈0001〉) and the second rotation ' /) () = 〈112 0〉).

Fig. 7

 7 Fig. 7 Micrographs of the α phase in the specimen deformed to the true strain of 0.29. (a) EBSD Y axis IPF micrograph; (b) KAM micrograph.

Fig. 8

 8 Fig. 8 EBSD Y axis IPF micrographs and the corresponding 0001 and 101 0 pole figures of the grains in two necklace regions. (a) Necklace region with less small grains representing the beginning of grain detachment; (b) necklace region with more small grains representing the mixing of small grains. The poles in the pole figures in (b) are from the coarse red grain and the small red domains.

Fig. 9

 9 Fig. 9 EBSD Y axis IPF micrograph of the specimen deformed to the true strain of 1.05.

  When the deformation progressed to the second stage, the formed symmetrical-tilt boundaries further evolved in two different ways. The one was by absorbing basal <a> dislocations formed due to incompatible deformation between neighboring grains in the bulged boundary regions, which transformed the symmetrical-tilt boundaries into asymmetrical-tilt boundaries characterized by a rotation around 〈101 〉. The other was by a rotational movement of the bulged parts around the normal axis of the symmetrical-tilt boundary (rotational sliding), which transformed the symmetrical-tilt boundary into a tilt-twist boundary characterized by a rotation around 〈112 〉. This resulted in the formation of the subgrains from the bulged grain boundary regions. When the deformation progressed to the third stage, the formed subgrains are detached from the parent grains and mixed with the ones detached from the neighboring grains by boundary sliding. Such a nucleation process from subgrain formation is also different from the classical subgrain growth by boundary migration or subgrain growth by coalescence. The three processes repeatedly happened from grain boundary regions toward grain interiors until the whole initial microstructure was replaced by the recrystallized grains.

Fig. A2- 1

 1 Fig. A2-1 (a) Schema illustrating the decomposition of the low-angle boundary into two symmetrical tilt boundaries made of dislocation with two different Burgers vectors or into one symmetrical-tilt boundary and a rotational sliding around the axis normal to the extra-half plane of the dislocation of symmetrical-tilt boundary; (b) schema showing the corresponding crystallographic rotations.

'

  ( /〈0001〉 and ' /〈101 0〉, the total rotation axis always falls near the 〈101 〉 line (the thick black line in Fig.A2-2 (a)) that runs from 〈0001〉 to 〈101 0〉. Similar to the case of 〈101 〉, when ) = 〈112 0〉 the combination of the two rotation angles also have a symmetrical contribution with respect to ' ( /' = 1 to the final disorientation angle, as shown in Fig. Fig. A2-2 (b). For the rotation axis, whatever the two rotations ' ( /〈0001〉 and ' /〈112 0〉, the final rotation axis always fall near the 〈112 〉 line (the thick black line in Fig. A2-2 (b)) that runs from 〈0001〉 to 〈112 0〉.

Fig. A2- 2

 2 Fig. A2-2 Calculated disorientation of low-angle boundary, in terms of θ/T on function of the first rotation ' ( /) ( () ( = 〈0001〉) and the second rotation ' /) . (a) ) = 〈101 0〉; (b) ) = 〈112 0〉.

Figure captions:Fig. 1

 1 Figure captions:

Fig. 2

 2 Fig. 2 Deformed high-temperature α microstructure at true strain of 0.05. (a1) EBSD Y axis

Fig. 3

 3 Fig. 3 (a) EBSD Y axis IPF micrograph of a representative example of boundary bulge

Fig. 4

 4 Fig. 4 Schema illustrating the formation of boundary bulging by dislocation slip and the

Fig. 5

 5 Fig. 5 Microstructural and crystallographic features of α grains in specimen deformed to true

Fig. 6

 6 Fig. 6 Schema illustrating the evolution of low-angle boundaries with further deformation. (a)

Fig. 7

 7 Fig. 7 Micrographs of the α phase in the specimen deformed to the true strain of 0.29. (a)

Fig. 8

 8 Fig. 8 EBSD Y axis IPF micrographs and the corresponding 0001 and 101 0 pole figures of the grains in two necklace regions. (a) Necklace region with less small grains representing

Fig. 9

 9 Fig. 9 EBSD Y axis IPF micrograph of the specimen deformed to the true strain of 1.05.

Fig. A1

 A1 Fig. A1 Original EBSD micrographs with band quality indexed contrast of the specimens

Fig. A2- 1

 1 Fig. A2-1 (a) Schema illustrating the decomposition of the low-angle boundary into two

Fig. A2- 2

 2 Fig. A2-2 Calculated disorientation of low-angle boundary, in terms of θ/T on function of the

Table 1 α

 1 grain size and area fractions of grains of interest of the specimens with different true strains

True strain ε Retained coarse grains Newly-formed grains

	3.2.4 Continuous dynamic recrystallization (CDRX)
	3.2.4.1 Necklace structure
	Fig.
		Average size (μm)	Fraction (%)	Average size (μm)	Fraction (%)
	0	220±84.0	100	/	0
	0.05	326±160.2	100	/	0
	0.07	580±216.3	95.5	12±6.3	4.5
	0.29	490±284.5	64.4	26±11.4	35.6
	1.05	/	0	45±23.9	100

Table A2 -

 A2 1 Possible slip modes in disordered α-Ti3Al (c/a=1.60) and the corresponding Taylor axes

	Slip mode	Slip type	Taylor axis
	11 00 〈112 0〉 0001 〈112 0〉 101 1 〈12 10〉 101 1 〈112 3 〉 112 2 〈112 3 〉	Prismatic 〈<〉 Basal 〈<〉 Pyramidal 〈<〉 Pyramidal-Ⅰ 〈= + <〉 Pyramidal-Ⅱ 〈= + <〉	〈0001 〉 〈11 00〉 〈1 012〉 〈27 44 17 10〉 〈11 00〉

Table A2 -

 A2 2 Slip modes in disordered α-Ti3Al (c/a=1.60) and the axis normal to the extra-half plane of dislocation For the first case, the low-angle boundaries with disorientation axes of 〈101 〉, ) = 〈101 0〉 provides the smallest deviation (∆') of the calculated G ('/) from the experimental G ex ('/) ex . The results of several examples are displayed in Table A2-3. For the second case, the low-angle boundaries with disorientation axis of 〈112 〉, T2 = >112 0? offers the smallest ∆'. The results of several examples are displayed in Table A2-4.

	Slip mode	Extra half plane / Symmetrical-tilt boundary plane	Rotation axis of grain boundary sliding
	11 00 〈112 0〉 0001 〈112 0〉 101 1 〈12 10〉 101 1 〈112 3 〉 112 2 〈112 3 〉	112 0 112 0 12 10 5 5 10 26 5 5 10 26	〈112 0〉 〈112 0〉 〈12 10〉 〈112 3 〉 〈112 3 〉

Table A2 -

 A2 [START_REF] Sakai | Dislocation substructures developed during dynamic recrystallisation in polycrystalline nickel[END_REF] Calculation results for low-angle boundaries with disorientation axis around 〈101 〉.

	6.37°/>1 014 ? 12.06°/>1 103? 7.97°/>01 13 ? 13.37°/>1 013 ? 6.05°/>01 12? 6.29°/>022 1? 5.03°/>01 13? 6.60°/>1 011 ? 6.29°/>101 2 ? 13.99°/>1 011?	〈0001〉 〈101 0〉	5.6° 9.8° 6.5° 10.8° 7.8° 3.0° 7° 4.7° 4.1° 4.4° 1.4° 6.1° 4.1° 2.9° 2.8° 6.0° 4.3° 4.6° 5.8° 12.7°	6.3524°/ C0.4579 0.0266 0.4845 1.9040 D 12.0383°/ C0.5505 0.6079 0.0574 1.7564D 8.0197°/ >0.0383 0.6037 0.5654 1.7499 ? 13.3154°/ C0.5514 0.0637 0.6150 1.7495 D 6.0135°/ C0.0302 0.7152 0.7454 1.4722D 6.2584°/ >0.0137 0.9663 0.9801 0.4828? 5.0216°/ C0.0238 0.5645 0.5883 1.7634D 6.6206°/ C0.8920 0.0255 0.9175 0.9128 D 6.2960°/ C0.7134 0.0316 0.7450 1.4746 D 13.9568°/ >0.9344 0.0531 0.8813 0.8941?	0.1502 0.6018 0.2703 0.7373 0.1637 0.0818 0.1141 0.1494 0.1760 0.6480
	Experimental result					Calculated result
	G ex (@/ ex	A	B	@ A	@ B	G (@/	∆@

Table A2 -

 A2 [START_REF] Cram | Modelling discontinuous dynamic recrystallization using a physically based model for nucleation[END_REF] Calculation results for low-angle boundaries with disorientation axis around 〈112 〉.

	Experimental result			Calculated result
	G ex (@/ ex 5.53°/>1 21 3 ? 10.38°/>112 6? 5.46°/>1 21 2? 3.07°/>1 1 24 ? 10.36°/>21 1 2? 10.52°/>112 1?	A 〈0001〉 〈112 0〉 B	@ A 3.5° 8.8° 2.6° 2.2° 4.9° 3.1° 10.1° @ B 4.3° 5.5° 4.8° 2.1° 9.1°	G (@/ 5.5438°/ >0.4707 0.8940 0.4234 1.3632 ? 10.3745°/ >0.3457 0.2644 0.6101 1.8310? 5.4586°/ C0.4869 1.0137 0.5268 1.0284D 3.0413°/ C0.3848 0.4113 0.7960 1.5625 D 10.3329°/ C1.0148 0.5450 0.4698 1.0223D 10.5639°/ 〈0.5252 0.5769 1.1021 0.6323〉	∆@ 0.1391 0.4220 0.1137 0.0573 0.3914 0.4717

Acknowledgements

This work was supported by the National Natural Science Foundation of China under grant No. 51571162, and the Research Found of the State Key Laboratory of Solidification Processing (NWPU), China under grant No. 2020-TZ-03. The first author Fengming Qiang is grateful to the China Scholarship Council for the support to her PhD study in France. The authors acknowledge the inventors of the Merengue 2 for the reconstruction of hightemperature α phase in the present work.

Appendix 2: Extended Intragranular Misorientation Axis (IGMA) analysis

In this appendix, an extension of the Intragranular Misorientation Axis (IGMA) analysis to complex cases is presented where different slip modes or even slip and grain boundary sliding are both activated in a grain. The IGMA analysis allows to determine the dominant slip system inside a grain by matching the theoretical rotation axis (Taylor axis) for a given slip system to those of the geometrically necessary dislocation (GND) forming the low-angle boundaries in a deformed grain. This analysis method works well for hexagonal materials of which only a few slip systems are available and often one is dominant. The theoretical Taylor axes have been calculated for different slip modes in hexagonal materials (see Table A2-1).

Below, the analysis method is extended, in addition to the primary slip mode, to identify a secondary slip mode or a rotational grain boundary sliding.

We assume that the disorientation ('/): ' rotation angle and ) rotation axis) associated to a low-angle boundary constituted of dislocations, can be decomposed into several single or basic disorientations each characterized by a specific rotation axis. The basic disorientations are either from symmetrical-tilt boundaries made of dislocations with the same Burgers vector 29 and the rotation axis is the so-called Taylor axis, as listed in Table A2-1, or from rotational boundary sliding around the axis normal to the boundary, as listed in Table A2-2. For clarity and simplicity, we suppose that '/) is composed of two single rotations, ' ( /) ( and ' /) (Fig. A2-1 (a)). Thus the following relation in matrix notation of the rotations holds:

where ,, , ( and , are the rotational matrices representing the misorientation of '/), ' ( /) ( and ' /) , as illustrated with the flow chart in Fig. A2-1 (b), and -. and -0 the i th and j th rotational symmetry matrices of the crystal structure. The rotational matrix of , can be calculated using the rotation angle ' and the unit rotation axis ) 1 ( , 1 , 1 2 as follows:

1 -1 2 cos ' + 1 2 :, (A2-2)

By knowing any of two rotations, the remaining rotation can be resolved. This formulation can be readily extended to a rotation composed of multiple single rotations.