Selective brain cooling achieves peripheral organs protection in hemorrhagic shock resuscitation via preserving the integrity of the brain-gut axis.

Background: Although whole-body cooling has been reported to improve the ischemic/reperfusion injury in hemorrhagic shock (HS) resuscitation, it is limited by its adverse reactions following therapeutic hypothermia. HS affects the experimental and clinical bowel disorders via activation of the brain-gut axis. It is unknown whether selective brain cooling achieves beneficial effects in HS resuscitation via preserving the integrity of the brain-gut axis. Methods: Male Sprague-Dawley rats were bled to hypovolemic HS and resuscitated with blood transfusion followed by retrograde jugular vein flush (RJVF) with 4 °C or 36 °C normal saline. The mean arterial blood pressure, cerebral blood flow, and brain and core temperature were measured. The integrity of intestinal tight junction proteins and permeability, blood pro-inflammatory cytokines, and multiple organs damage score were determined. Results: Following blood transfusion resuscitation, HS rats displayed gut barrier disruption, increased blood levels of pro-inflammatory cytokines, and peripheral vital organ injuries. Intrajugular-based infusion cooled the brain robustly with a minimal effect on body temperature. This brain cooling significantly reduced the HS resuscitation-induced gut disruption, systemic inflammation, and peripheral vital organ injuries in rats. Conclusion: Resuscitation with selective brain cooling achieves peripheral vital organs protection in hemorrhagic shock resuscitation via preserving the integrity of the brain-gut axis.

Heat Shock Protein 70 (HSP70) Reduces Hepatic Inflammatory and Oxidative Damage in a Rat Model of Liver Ischemia/Reperfusion Injury with Hyperbaric Oxygen Preconditioning.

BACKGROUND Several clinical conditions can cause hepatic ischemia/reperfusion (I/R) injury. This study aimed to determine the mechanism of the protective effect of hyperbaric oxygen preconditioning (HBO2P) on hepatic ischemia/reperfusion (I/R) injury in a rat model, and to investigate the effects on HBO2P and I/R injury of blocking HSP70 using antibody (Ab) pretreatment. MATERIAL AND METHODS Male Sprague-Dawley rats underwent HBO2P for 60 min at 2.0 atmosphere absolute (ATA) pressure for five consecutive days before surgical hepatic I/R injury, performed by clamping the portal vein and hepatic lobe. Four groups studied included: the non-HBO2P+ non-I/R group, which underwent sham surgery (N=10); the non-HBO2P + I/R group (N=10); the HBO2P + I/R group (N=10); and the HBO2P + HSP70-Ab + I/R group (N=10) received one dose of HSP70 antibody one day before hepatic I/R injury. Serum lactate dehydrogenase (LDH), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and pro-inflammatory cytokines, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), and hepatic malondialdehyde (MDA) and myeloperoxidase (MPO) were measured biochemically. Rat liver tissues were examined histologically. RESULTS In rats with hepatic I/R injury without HSP70 antibody pre-treatment, HBO2P significantly reduced hepatic injury and levels of LDH, AST, ALT, TNF-α, IL-6, MDA, and MPO levels; in comparison, the group pre-treated with an antibody to inhibit HSP70 (the HBO2P + HSP70-Ab + I/R group) showed significant reversal of the beneficial effects of HBO2P on hepatic I/R injury (p<0.05). CONCLUSIONS In a rat model of hepatic I/R injury with HBO2P, HSP70 reduced hepatic inflammatory and oxidative damage.

Gamma-Secretase Inhibitors Attenuate Neurotrauma and Neurogenic Acute Lung Injury in Rats by Rescuing the Accumulation of Hypertrophic Microglia.

BACKGROUND/AIMS: In response to traumatic brain injury (TBI), activated microglia exhibit changes in their morphology from the resting ramified phenotype toward the activated hypertrophic or amoeboid phenotype. Here, we provide the first description of the mechanism underlying the neuroprotective effects of γ-secretase inhibitors on TBI outcomes in rats. METHODS: The neuroprotective effects of γ-secretase inhibitors such as LY411575 or CHF5074 on TBI-induced neurotoxicity were analysed using a neurological motor function evaluation, cerebral contusion assay, immunohistochemical staining for microglia phenotypes, lung injury score and Evans Blue dye extravasation assay of brain and lung oedema. RESULTS: Hypertrophic or amoeboid microglia accumulated in the injured cortex, the blood-brain-barrier was disrupted and neurological deficits and acute lung injury were observed 4 days after TBI in adult rats. However, a subcutaneous injection of LY411575 (5 mg/kg) or CHF5074 (30 mg/kg) immediately after TBI and once daily for 3 consecutive days post-TBI significantly attenutaed the accumulation of hypertrophic microglia in the injured brain, neurological injury, and neurogenic acute lung injury. CONCLUSION: Gamma-secretase inhibitors attenuated neurotrauma and neurogenic acute lung injury in rats by reducing the accumulation of hypertrophic microglia in the vicinity of the lesion.

Exercise attenuates neurological deficits by stimulating a critical HSP70/NF-κB/IL-6/synapsin I axis in traumatic brain injury rats.

BACKGROUND: Despite previous evidence for a potent inflammatory response after a traumatic brain injury (TBI), it is unknown whether exercise preconditioning (EP) improves outcomes after a TBI by modulating inflammatory responses. METHODS: We performed quantitative real-time PCR (qPCR) to quantify the genes encoding 84 cytokines and chemokines in the peripheral blood and used ELISA to determine both the cerebral and blood levels of interleukin-6 (IL-6). We also performed the chromatin immunoprecipitation (ChIP) assay to evaluate the extent of nuclear factor kappa-B (NF-κB) binding to the DNA elements in the IL-6 promoter regions. Also, we adopted the Western blotting assay to measure the cerebral levels of heat shock protein (HSP) 70, synapsin I, and ß-actin. Finally, we performed both histoimmunological and behavioral assessment to measure brain injury and neurological deficits, respectively. RESULTS: We first demonstrated that TBI upregulated nine pro-inflammatory and/or neurodegenerative messenger RNAs (mRNAs) in the peripheral blood such as CXCL10, IL-18, IL-16, Cd-70, Mif, Ppbp, Ltd, Tnfrsf 11b, and Faslg. In addition to causing neurological injuries, TBI also upregulated the following 14 anti-inflammatory and/or neuroregenerative mRNAs in the peripheral blood such as Ccl19, Ccl3, Cxcl19, IL-10, IL-22, IL-6, Bmp6, Ccl22, IL-7, Bmp7, Ccl2, Ccl17, IL-1rn, and Gpi. Second, we observed that EP inhibited both neurological injuries and six pro-inflammatory and/or neurodegenerative genes (Cxcl10, IL-18, IL-16, Cd70, Mif, and Faslg) but potentiated four anti-inflammatory and/or neuroregenerative genes (Bmp6, IL-10, IL-22, and IL-6). Prior depletion of cerebral HSP70 with gene silence significantly reversed the beneficial effects of EP in reducing neurological injuries and altered gene profiles after a TBI. A positive Pearson correlation exists between IL-6 and HSP70 in the peripheral blood or in the cerebral levels. In addition, gene silence of cerebral HSP70 significantly reduced the overexpression of NF-κB, IL-6, and synapsin I in the ipsilateral brain regions after an EP in rats. CONCLUSIONS: TBI causes neurological deficits associated with stimulating several pro-inflammatory gene profiles but inhibiting several anti-inflammatory gene profiles of cytokines and chemokines. Exercise protects against neurological injuries via stimulating an anti-inflammatory HSP70/NF-κB/IL-6/synapsin I axis in the injured brains.

Combined Hemorrhagic Shock and Unilateral Common Carotid Occlusion Induces Neurological Injury in Adult Male Rats.

Background: Clinical assessment reveals that patients after surgery of cardiopulmonary bypass or coronary bypass experience postoperative cognitive dysfunction. This study aimed to investigate whether resuscitation after a hemorrhagic shock (HS) and/or mild cerebral ischemia caused by a unilateral common carotid artery occlusion (UCCAO) can cause brain injury and concomitant neurological dysfunction, and explore the potential mechanisms. Methods: Blood withdrawal (6 mL/100 g body weight) for 60 min through the right jugular vein catheter-induced an HS. Immediately after the termination of HS, we reinfused the initially shed blood volumes to restore and maintain the mean arterial blood pressure (MABP) to the original value during the 30-min resuscitation. A cooling water blanket used to induce whole body cooling for 30 min after the end of resuscitation. Results: An UCCAO caused a slight cerebral ischemia (cerebral blood flow [CBF] 70%) without hypotension (MABP 85 mmHg), systemic inflammation, multiple organs injuries, or neurological injury. An HS caused a moderate cerebral ischemia (52% of the original CBF levels), a moderate hypotension (MABP downed to 22 mmHg), systemic inflammation, and peripheral organs injuries. However, combined an UCCAO and an HS caused a severe cerebral ischemia (18% of the original CBF levels), a moderate hypotension (MABP downed to 17 mmHg), systemic inflammation, peripheral organs damage, and neurological injury, which can be attenuated by whole body cooling. Conclusions: When combined with an HS, an UCCAO is associated with ischemic neuronal injury in the ipsilateral hemisphere of adult rat brain, which can be attenuated by therapeutic hypothermia. A resuscitation from an HS regards as a reperfusion insult which may induce neurological injury in patients with an UCCAO disease.

A positive correlation exists between neurotrauma and TGF-ß1-containing microglia in rats.

BACKGROUND: Transforming growth factor-beta 1 (TGF-ß1) regulates many processes after traumatic brain injury (TBI). Both Neuro AiD™ (MLC601) and astragaloside (AST) attenuate microglia activation in rats with TBI. The purpose of this study was to investigate whether MLC601 or AST improves output of TBI by affecting microglial expression of TGF-ß1. MATERIALS AND METHODS: Adult male Sprague-Dawley rats (120 in number) were used to investigate the contribution of TGF-ß1-containing microglia in the MLC601-mediated or the AST-mediated neuroprotection in the brain trauma condition using lateral fluid percussion injury. RESULTS: Pearson correlation analysis revealed that there was a positive correlation between brain injury (evidenced by both brain contused volume and neurological severity score) and the cortical numbers of TGF-ß1-containing microglia for the rats (n = 12) 4 days post-TBI. MLC601 or AST significantly (P < 0·05) attenuated TBI-induced brain contused volume (119 ± 14 mm3 or 108 ± 11 mm3 vs. 160 ± 21 mm3 ), neurological severity score (7·8 ± 0·3 or 8·1 ± 0·4 vs. 10·2 ± 0·5) and numbers of TGF-ß1-containing microglia (6% ± 2% or 11% ± 3% vs. 79% ± 7%) for the rats 4 days post-TBI. CONCLUSIONS: There was a positive correlation between TBI and cortical numbers of TGF-ß1-containing microglia which could be significantly attenuated by astragaloside or NeuroAiD™ (MLC601) in rats.

Proteomic analysis of hypothalamic injury in heatstroke rats.

Ischemic and oxidative damage to the hypothalamus may be associated with decreased heat tolerance as well as heatstroke formation. The present study explores the hypothalamic proteome mechanisms associated with heatstroke-mediated hypothalamic ischemia, and oxidative damage. Heatstroke rats had hypotension, hypothalamic ischemia, and lethality. In addition, they had hyperthermia and hypothalamic blood-brain-barrier disruption, oxidative stress, activated inflammation, and neuronal apoptosis and degeneration. 2DE combined LC-MS/MS revealed that heatstroke-induced ischemic injury and apoptosis were associated with upregulation of L-lactate dehydrogenase but downregulation of both dihydropyriminase-related protein and 14-3-3 Zeta isoform protein. Heat-induced blood-brain-barrier disruption might be related to upregulation of glial fibrillary acidic protein. Oxidative stress caused by heatstroke might be related to upregulation of cytosolic dehydrogenase-1. Also, heat-induced overproduction of proinflammatory cytokines might be associated with downregulation of stathmin 1. Heat-induced hypothalamic ischemia, apoptosis, injury (or upregulation of L-lactate dehydrogenase), blood-brain-barrier disruption (or upregulation of glial fibrillary acidic protein), oxidative stress (or upregulation of cytosolic dehydrogenase-1), and activated inflammation (or downregulation of stathmin 1) were all significantly reversed by whole body cooling. Our data indicate that cooling therapy improves outcomes of heatstroke by modulating hypothalamic proteome mechanisms.

Protecting against ischaemic stroke in rats by heat shock protein 20-mediated exercise.

BACKGROUND: Exercise preconditioning (EP(+) ) has been widely accepted as a being of safe and effective preventive measure for stroke. The purpose of this study was to investigate whether EP(+) improves outcomes of ischaemic stroke by promoting neuronal and glial expression of heat shock protein (HSP) 20. MATERIALS AND METHODS: Adult male Sprague-Dawley rats (288 in number) were used to investigate the contribution of HSP20-containing neurons and HSP20-containing glial cells in the exercise-mediated neuroprotection in the stroke condition using middle cerebral artery occlusion. RESULTS: Exercise preconditioning, in addition to increasing the numbers of both the HSP20-containg neurons (88 ± 8 vs. 43 ± 4; n = 8 each group; P < 0·05) and the HSP20-containg astrocytes (102 ± 10 vs. 56 ± 5; n = 8; P < 0·05) significantly attenuated stroke-induced brain infarct (140 ± 9 vs. 341 ± 20 mm(3) ; n = 8 per group; P < 0·01), neuronal apoptosis (20 ± 5 vs. 87 ± 7; n = 8 per group; n = 8; P < 0·01), glial apoptosis (29 ± 5 vs. 101 ± 4; n = 8; P < 0·01), and neurological deficits (6·6 ± 0·3 vs. 11·7 ± 0·8; n = 8 per group; P < 0·01). Reducing the numbers of both HSP20-containing neurons and HSP20-contaiing glia by intracerebral injection of pSUPER small interfering RNAί expressing HSP20 significantly reversed the beneficial effects of EP(+) in attenuating stroke-induced cerebral infarct, neuronal and glial apoptosis, and neurological deficits. CONCLUSIONS: The numbers of both the HSP20-containing neurons and the HSP20-containing glia inversely correlated with the outcomes of ischaemic stroke. In addition, preischaemic treadmill exercise improves outcomes of ischaemic stroke by increasing the numbers of both the HSP20-containing neurons and the HSP20-containing glia.

Attenuating systemic inflammatory markers in simulated high-altitude exposure by heat shock protein 70-mediated hypobaric hypoxia preconditioning in rats.

BACKGROUND/PURPOSE: The primary goal of this study was to test whether high-altitude exposure (HAE: 0.9% O(2) at 0.47 ATA for 24 hours) was capable of increasing the systemic inflammatory markers as well as the toxic organ injury indicators in rats, with a secondary goal to test whether preinduction of heat shock protein (HSP) 70 by hypobaric hypoxia preconditioning (HHP: 18.3% O(2) at 0.66 ATA for 5 h/day on 5 days consecutively for 2 weeks) attenuated the proposed increased serum levels of both the systemic inflammatory markers and the toxic organ injury indicators. METHODS: Rats were assigned to: (1) non-HHP (21% O(2) at 1.0 ATA)+non-HAE (21% O(2) at 1.0 ATA) group; (2) non-HHP+HAE group; (3) HHP+non-HAE group; (4) HHP+HAE group; and (5) HHP+HSP70 antibodies (Ab)+HAE group. For the HSP70Ab group, a neutralizing HSP70Ab was injected intravenously at 24 hours prior to HAE. All the physiological and biochemical parameters were obtained at the end of HAE or the equivalent time period of non-HAE. Blood samples were obtained for determination of both the systemic inflammatory markers (e.g., serum tumor necrosis factor-α, interleukin-1ß, E-selectin, intercellular adhesion molecule-1, and liver myeloperoxidase activity) and the toxic organ injury indicators (e.g., nitric oxide metabolites, 2,3-dihydroxybenzoic acid, and lactate dehydrogenase). RESULTS: HHP, in addition to inducing overexpression of tissue HSP70, significantly attenuated the HAE-induced hypotension, bradycardia, hypoxia, acidosis, and increased tissue levels of both the systemic inflammatory markers and the toxic organ injury indicators. The beneficial effects of HHP in inducing tissue overexpression of HSP70 as well as in preventing the HAE-induced increased levels of the systemic inflammatory markers and the toxic organ injury indicators could be significantly reduced by HSP70Ab preconditioning. CONCLUSION: These results suggest that HHP may downgrade both the systemic inflammatory markers and the toxic organ injury indicators in HAE by upregulating tissue HSP70.

Involvement of TG-interacting factor in microglial activation during experimental traumatic brain injury.

Traumatic brain injury (TBI) is a complex injury involving several physiological alterations, potentially leading to neurological impairment. Previous mouse studies using high-density oligonucleotide array analysis have confirmed the upregulation of transforming growth-interacting factor (TGIF) mRNA in TBI. TGIF is a transcriptional corepressor of transforming growth factor beta (TGF-ß) signaling which plays a protective role in TBI. However, the functional roles of TGIF in TBI are not well understood. In this study, we used confocal microscopy after immunofluorescence staining to demonstrate the increase of TGIF levels in the activated microglia of the pericontusional cortex of rats with TBI. Intracerebral knockdown of TGIF in the pericontusional cortex significantly downregulated TGIF expression, attenuated microglial activation, reduced the volume of damaged brain tissue, and facilitated recovery of limb motor function. Collectively, our results indicate that TGIF is involved in TBI-induced microglial activation, resulting in secondary brain injury and motor dysfunction. This study investigated the roles of transforming growth-interacting factor (TGIF) in a traumatic brain injury (TBI)-rat model. We demonstrated the increase of TGIF levels in the activated microglia of the pericontusional cortex of rats with TBI. Intracerebral knockdown of TGIF in the pericontusional cortex of the TBI rats significantly attenuated micoglial activation, reduced the volume of damaged brain tissue, and facilitated recovery of limb motor function. We suggest that inhibition of TGIF might provide a promising therapeutic strategy for TBI.

Beneficial effect of astragalosides on stroke condition using PC12 cells under oxygen glucose deprivation and reperfusion.

Astragalosides (AST) are reported to be neuroprotective in focal cerebral ischemic models in vivo. In this study, the direct effect of AST against oxygen and glucose deprivation (OGD) including neuronal injury and the underlying mechanisms in vitro were investigated. 5 h OGD followed by 24 h of reperfusion [adding back oxygen and glucose (OGD-R)] was used to induce in vitro ischemia reperfusion injury in differentiated rat pheochromocytoma PC12 cells. AST (1, 100, and 200 µg/mL) were added to the culture after 5 h of the OGD ischemic insult and was present during the reoxygenation phases. A key finding was that OGD-R decreased cell viability, increased lactate dehydrogenase, increased reactive oxygen species, apoptosis, autophagy, functional impairment of mitochondria, and endoplasmic reticulum stress in PC12 cells, all of which AST treatment significantly reduced. In addition, AST attenuated OGD-R-induced cell loss through P38 MAPK activation a neuroprotective effect blunted by SB203580, a specific inhibitor of P38 MAPK. Our data suggest that both apoptosis and autophagy are important characteristics of OGD-R-induced PC12 death and that treating PC12 cells with AST blocked OGD-R-induced apoptosis and autophagy by suppressing intracellular oxidative stress, functional impairment of mitochondria, and endoplasmic reticulum stress. Our data provide identification of AST that can concomitantly inhibit multiple cells death pathways following OGD injuries in neural cells.

Repetitive hyperbaric oxygen therapy provides better effects on brain inflammation and oxidative damage in rats with focal cerebral ischemia.

BACKGROUND/PURPOSE: Repetitive hyperbaric oxygen (HBO2) therapy may cause excessive generation of reactive oxygen species. This study assessed whether repetitive or 2-4-day trials of HBO2 therapy (2 treatments daily for 2-4 consecutive days) provides better effects in reducing brain inflammation and oxidative stress caused by middle cerebral artery occlusion (MCAO) in rats than did a 1-day trial of HBO2 therapy (2 treatments for 1 day). METHODS: Rats were randomly divided into four groups: sham; MCAO without HBO2 treatment; MCAO treated with 1-day trial of HBO2; and MCAO treated with 2-4-day trials of HBO2. One treatment of HBO2 (100% O2 at 253 kPa) lasted for 1 hour in a hyperbaric chamber. RESULTS: Therapy with the 2-4-day trials of HBO2 significantly and dose-dependently attenuated the MCAO-induced cerebral infarction and neurological deficits more than the 1-day trial of HBO2 therapy. The beneficial effects of repetitive HBO2 therapy were associated with: (1) reduced inflammatory status in ischemic brain tissues (evidenced by decreased levels of tumor necrosis factor-α, interleukin-1ß, and myeloperoxidase activity); (2) decreased oxidative damage in ischemic brain tissues (evidenced by decreased levels of reactive oxygen and nitrogen species, lipid peroxidation, and enzymatic pro-oxidants, but increased levels of enzymatic antioxidant defenses); and (3) increased production of an anti-inflammatory cytokine, interleukin-10. CONCLUSION: The results provide the apparently contradictory finding that heightened oxygen tension reduced oxidative stress (and inflammation), which was reflected by increased antioxidant and decreased oxidant contents under focal cerebral ischemia.

Exercise preconditioning protects against spinal cord injury in rats by upregulating neuronal and astroglial heat shock protein 72.

The heat shock protein 72 (HSP 72) is a universal marker of stress protein whose expression can be induced by physical exercise. Here we report that, in a localized model of spinal cord injury (SCI), exercised rats (given pre-SCI exercise) had significantly higher levels of neuronal and astroglial HSP 72, a lower functional deficit, fewer spinal cord contusions, and fewer apoptotic cells than did non-exercised rats. pSUPER plasmid expressing HSP 72 small interfering RNA (SiRNA-HSP 72) was injected into the injured spinal cords. In addition to reducing neuronal and astroglial HSP 72, the (SiRNA-HSP 72) significantly attenuated the beneficial effects of exercise preconditioning in reducing functional deficits as well as spinal cord contusion and apoptosis. Because exercise preconditioning induces increased neuronal and astroglial levels of HSP 72 in the gray matter of normal spinal cord tissue, exercise preconditioning promoted functional recovery in rats after SCI by upregulating neuronal and astroglial HSP 72 in the gray matter of the injured spinal cord. We reveal an important function of neuronal and astroglial HSP 72 in protecting neuronal and astroglial apoptosis in the injured spinal cord. We conclude that HSP 72-mediated exercise preconditioning is a promising strategy for facilitating functional recovery from SCI.

Mesenchymal stem cells reduce long-term cognitive deficits and attenuate myelin disintegration and microglia activation following repetitive traumatic brain injury.

The underlying mechanisms for the beneficial effects exerted by bone marrow-mesenchymal stem cells (BM-MSCs) in treating repetitive traumatic brain injury (rTBI)-induced long-term sensorimotor/cognitive impairments are not fully elucidated. Herein, we aimed to explore whether BM-MSCs therapy protects against rTBI-induced long-term neurobehavioral disorders in rats via normalizing white matter integrity and gray matter microglial response. Rats were subjected to repeated mild lateral fluid percussion on day 0 and day 3. On the fourth day post-surgery, MSCs groups received MSCs (4 × 106 cells/ml/kg, intravenously) and were assessed by the radial maze, Y maze, passive avoidance tests, and modified neurological severity scores. Hematoxylin & eosin, and Luxol fast blue stainings were used to examine the histopathology and white matter thickness. At the same time, immunofluorescence staining was used to investigate the numbers of tumor necrosis factor-alpha (TNF-α)-containing microglia in gray matter. Three to nine months after neurotrauma, rats displayed sensorimotor and cognitive impairments, reduced thickness in white matter, and over-accumulation of TNF-α-containing microglia and cellular damage in gray matter. Therapy with BM-MSCs significantly attenuated the rTBI-induced sensorimotor and cognitive impairments and all their complications. Mesenchymal stem cell therapy might accelerate the recovery of sensorimotor and cognitive impairments in rats with rTBI via normalizing myelin integrity and microglia response.

White and gray matter integrity evaluated by MRI-DTI can serve as noninvasive and reliable indicators of structural and functional alterations in chronic neurotrauma.

We aimed to evaluate whether white and gray matter microstructure changes observed with magnetic resonance imaging (MRI)-based diffusion tensor imaging (DTI) can be used to reflect the progression of chronic brain trauma. The MRI-DTI parameters, neuropathologic changes, and behavioral performance of adult male Wistar rats that underwent moderate (2.1 atm on day "0") or repeated mild (1.5 atm on days "0" and "2") traumatic brain injury (TBI or rmTBI) or sham operation were evaluated at 7 days, 14 days, and 1-9 months after surgery. Neurobehavioral tests showed that TBI causes long-term motor, cognitive and neurological deficits, whereas rmTBI results in more significant deficits in these paradigms. Both histology and MRI show that rmTBI causes more significant changes in brain lesion volumes than TBI. In vivo DTI further reveals that TBI and rmTBI cause persistent microstructural changes in white matter tracts (such as the body of the corpus callosum, splenium of corpus callus, internal capsule and/or angular bundle) of both two hemispheres. Luxol fast blue measurements reveal similar myelin loss (as well as reduction in white matter thickness) in ipsilateral and contralateral hemispheres as observed by DTI analysis in injured rats. These data indicate that the disintegration of microstructural changes in white and gray matter parameters analyzed by MRI-DTI can serve as noninvasive and reliable markers of structural and functional level alterations in chronic TBI.

Hypoxic preconditioning enhances the therapeutic potential of the secretome from cultured human mesenchymal stem cells in experimental traumatic brain injury.

Bone-marrow-derived human MSCs (mesenchymal stem cells) support repair when administered to animals with TBI (traumatic brain injury) in large part through secreted trophic factors. We directly tested the ability of the culture medium (or secretome) collected from human MSCs under normoxic or hypoxic conditions to protect neurons in a rat model of TBI. Concentrated conditioned medium from cultured human MSCs or control medium was infused through the tail vein of rats subjected to TBI. We have demonstrated that MSCs cultured in hypoxia were superior to those cultured in normoxia in inducing expression of both HGF (hepatocyte growth factor) and VEGF (vascular endothelial growth factor) in the cultured medium. We showed further that rats treated with the secretome from both normoxic- and hypoxic-preconditioned MSCs performed significantly better than the controls in both motor and cognitive functional test. Subsequent post-mortem evaluation of brain damage at the 4-day time point confirmed that both normoxic- and hypoxic-preconditioned MSC secretome-treated rats had significantly greater numbers of newly forming neurons, but significantly less than the controls in brain damaged volume and apoptosis. The TBI rats treated with hypoxic-preconditioned MSC secretome performed significantly better in both motor and cognitive function tests and neurogenesis, and had significantly less brain damage than the TBI rats treated with the normoxic-preconditioned MSC secretome. Collectively, these findings suggest that MSCs secrete bioactive factors, including HGF and VEGF, that stimulate neurogenesis and improve outcomes of TBI in a rat model. Hypoxic preconditioning enhances the secretion of these bioactive factors from the MSCs and the therapeutic potential of the cultured MSC secretome in experimental TBI.

Attenuating heat-induced cellular autophagy, apoptosis and damage in H9c2 cardiomyocytes by pre-inducing HSP70 with heat shock preconditioning.

PURPOSE: We sought to assess whether heat-induced autophagy, apoptosis and cell damage in H9c2 cells can be affected by pre-inducing HSP70 (heat shock protein 70). MATERIALS AND METHODS: Cell viability was determined using 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide staining and a lactate dehydrogenase assay. Apoptosis was evidenced using both flow cytometry and counting caspase-3 positive cells, whereas autophagy was evidenced by the increased LC3-II expression and lysosomal activity. RESULTS: The viability of H9c2 cells was temperature-dependently (40-44 °C) and time-dependently (90-180 min) significantly (p < 0.05) reduced by severe heat, which caused cell damage, apoptosis and autophagy. Heat-induced cell injury could be attenuated by pretreatment with 3-methylademine (an autophagy inhibitor) or Z-DEVD-FMK (a caspase-3 inhibitor). Neither apoptosis nor autophagy over the levels found in normothermic controls was induced in heat-shock preconditioned controls (no subsequent heat injury). The beneficial effects of mild heat preconditioning (preventing heat-induced cell damage, apoptosis and autophagy) were significantly attenuated by inhibiting HSP70 overexpression with triptolide (Tripterygium wilfordii) pretreatment. CONCLUSION: We conclude that pre-inducing HSP70 attenuates heat-stimulated cell autophagy, apoptosis and damage in the heart. However, this requires in vivo confirmation.

Ischemic and oxidative damage to the hypothalamus may be responsible for heat stroke.

The hypothalamus may be involved in regulating homeostasis, motivation, and emotional behavior by controlling autonomic and endocrine activity. The hypothalamus communicates input from the thalamus to the pituitary gland, reticular activating substance, limbic system, and neocortex. This allows the output of pituitary hormones to respond to changes in autonomic nervous system activity. Environmental heat stress increases cutaneous blood flow and metabolism, and progressively decreases splanchnic blood flow. Severe heat exposure also decreases mean arterial pressure (MAP), increases intracranial pressure (ICP), and decreases cerebral perfusion pressure (CPP = MAP - ICP), all of which lead to cerebral ischemia and hypoxia. Compared with normothermic controls, rodents with heatstroke have higher hypothalamic values of cellular ischemia (e.g., glutamate and lactate-to-pyruvate ratio) and damage (e.g., glycerol) markers, pro-oxidant enzymes (e.g., lipid peroxidation and glutathione oxidation), proinflammatory cytokines (e.g., interleukin-1ß and tumor necrosis factor-α), inducible nitric oxide synthase-dependent nitric oxide, and an indicator for the accumulation of polymorphonuclear leukocytes (e.g., myeloperoxidase activity), as well as neuronal damage (e.g., apoptosis, necrosis, and autophagy) after heatstroke. Hypothalamic values of antioxidant defenses (e.g., glutathione peroxidase and glutathione reductase), however, are lower. The ischemic, hypoxic, and oxidative damage to the hypothalamus during heatstroke may cause multiple organ dysfunction or failure through hypothalamic-pituitary-adrenal axis mechanisms. Finding the link between the signaling and heatstroke-induced hypothalamic oxidative and ischemic damage might allow us to clinically attenuate heatstroke. In particular, free radical scavengers, heat shock protein-70 inducers, hypervolemic hemodilution, inducible nitric oxide synthase inhibitors, progenitor stem cells, flutamide, estrogen, interleukin-1 receptor antagonists, glucocorticoid, activated protein C, and baicalin mitigate preclinical heatstroke levels.

Melatonin improves outcomes of heatstroke in mice by reducing brain inflammation and oxidative damage and multiple organ dysfunction.

We report here that when untreated mice underwent heat stress, they displayed thermoregulatory deficit (e.g., animals display hypothermia during room temperature exposure), brain (or hypothalamic) inflammation, ischemia, oxidative damage, hypothalamic-pituitary-adrenal axis impairment (e.g., decreased plasma levels of both adrenocorticotrophic hormone and corticosterone during heat stress), multiple organ dysfunction or failure, and lethality. Melatonin therapy significantly reduced the thermoregulatory deficit, brain inflammation, ischemia, oxidative damage, hypothalamic-pituitary-adrenal axis impairment, multiple organ dysfunction, and lethality caused by heat stroke. Our data indicate that melatonin may improve outcomes of heat stroke by reducing brain inflammation, oxidative damage, and multiple organ dysfunction.

Attenuating brain inflammation, ischemia, and oxidative damage by hyperbaric oxygen in diabetic rats after heat stroke.

BACKGROUND/PURPOSE: Alternating hypothalamic-pituitary-adrenal axis mechanisms would lead to multiple organs dysfunction or failure. Herein, we attempt to assess whether hypothalamic inflammation and ischemic and oxidative damage that occurred during heatstroke (HS) can be affected by hyperbaric oxygen (HBO2) therapy in streptozotocin-induced diabetic rats. METHODS: In this study, anesthetized diabetic rats, immediately after the onset of HS, were divided into two major groups and given the normobaric air (21% O2 at 1.0 atmospheres absolute) or HBO2 (100% O2 at 2.0 atmospheres absolute). HS was induced by exposing the animals to heat stress (43°C). Another group of anesthetized diabetic rats was kept at normothermic state and used as controls. RESULTS: The survival time values for the HBO2-treated HS-diabetic rats increased form the control values of 78-82 minutes to new values of 184-208 minutes. HBO2 therapy caused a reduction of HS-induced cellular ischemia (e.g., increased cellular levels of glutamate and lactate/pyruvate ratio), hypoxia (e.g., decreased cellular levels of PO2), inflammation (e.g., increased cellular levels of interleukin-1ß, tumor necrosis factor-alpha, interleukin-6, and myeloperoxidase), and oxidative damage (e.g., increased values of nitric oxide, 2,3-dihydroxybenzoic acid, glycerol, and neuronal damage score) in the hypothalamus of the diabetic rats. CONCLUSION: Our results suggest that, in diabetic animals, HBO2 therapy may improve outcomes of HS in part by reducing heat-induced activated inflammation and ischemic and oxidative damage in the hypothalamus and other brain regions.