The adverse pial arteriolar and axonal consequences of traumatic brain injury complicated by hypoxia and their therapeutic modulation with hypothermia in rat

This study examined the effect of posttraumatic hypoxia on cerebral vascular responsivity and axonal damage, while also exploring hypothermia''s potential to attenuate these responses. Rats were subjected to impact acceleration injury (IAI) and equipped with cranial windows to assess vascular reactivity to topical acetylcholine, with postmortem analyses using antibodies to amyloid precursor protein to assess axonal damage. Animals were subjected to hypoxia alone, IAI and hypoxia, IAI and hypoxia before induction of moderate hypothermia (33°C), IAI and hypoxia induced during hypothermic intervention, and IAI and hypoxia initiated after hypothermia. Hypoxia alone had no impact on vascular reactivity or axonal damage. Acceleration injury and posttraumatic hypoxia resulted in dramatic axonal damage and altered vascular reactivity. When IAI and hypoxia were followed by hypothermic intervention, no axonal or vascular protection ensued. However, when IAI was followed by hypoxia induced during hypothermia, axonal and vascular protection followed. When this same hypoxic insult followed the use of hypothermia, no benefit ensued. These studies show that early hypoxia and delayed hypoxia exert damaging axonal and vascular consequences. Although this damage is attenuated by hypothermia, this follows only when hypoxia occurs during hypothermia, with no benefit found if the hypoxic insult proceeds or follows hypothermia.     

Bimodal upregulation of glial cell line-derived neurotrophic factor (GDNF) in the neonatal rat brain following ischemic/hypoxic injury

In order to delineate the spatial and temporal patterns of glial cell line-derived neurotrophic factor (GDNF) expression following ischemic/hypoxic injury in immature and neonatal brain, GDNF protein levels and immunocytochemistry were studied in rats subjected to a modified Levine procedure. Significant upregulation of GDNF protein occurred in a bimodal fashion in the damaged left cerebral cortex and hippocampus, while the levels in the right cerebral hemisphere of both control and ischemic groups remained relatively unchanged. Immunocytochemical studies indicated that the early rise in GDNF levels was most likely to be related to enhanced neuronal release of GDNF. The second rise was probably related to progressive astrogliosis that occurred in response to injury. In contrast to the lack of GDNF expression among astrocytes in normal mature brains, reactive astrocytes in the neonate appear to possess a ready capacity to express GDNF. Spatial and temporal changes in the pattern of GDNF expression following injury, as determined in this study may provide insight into the functions of GDNF in vivo and into possible therapeutic approaches toward prevention of damage or rescue of neurons following brain injury.     

Potentiation of intracellular Ca2+ mobilization by hypoxia-induced NO generation in rat brain striatal slices and human astrocytoma U-373 MG cells and its involvement in tissue damage

The relationship between nitric oxide (NO) and intracellular Ca2+ in hypoxic-ischemic brain damage is not known in detail. Here we used rat striatal slices perfused under low-oxygen and Ca2+-free conditions and cultured human astrocytoma cells incubated under similar conditions as models to study the dynamics of intracellular NO and Ca2+ in hypoxia-induced tissue damage. Exposure of rat striatal slices for 70 min to low oxygen tension elicited a delayed and sustained increase in the release of 45Ca2+. This was potentiated by the NO donors sodium nitroprusside (SNP) and spermine-NO and inhibited by N-omega-nitro-L-arginine methyl ester (L-NAME) or by the NO scavenger 2-phenyl-4,4,5,5 tetramethylimidazoline-1-oxyl-3-oxide (PTIO). A membrane-permeant form of heparin in combination with either ruthenium red (RR) or ryanodine (RY) also inhibited 45Ca2+ release. In human astrocytoma U-373 MG cells, hypoxia increased intracellular Ca2+ concentration ([Ca2+]i) by 67.2 +/- 13.1% compared to normoxic controls and this effect was inhibited by L-NAME, PTIO or heparin plus RR. In striatal tissue, hypoxia increased NO production and LDH release and both effects were antagonized by L-NAME. Although heparin plus RR or RY antagonized hypoxia-induced increase in LDH release they failed to counteract increased NO production. These data therefore indicate that NO contributes to hypoxic damage through increased intracellular Ca2+ mobilization from endoplasmic reticulum and suggest that the NO-Ca2+ signalling might be a potential therapeutic target in hypoxia-induced neuronal degeneration.     

A qualitative comparison of the glucocorticoid receptor in cytosol from human brain and rat brain

The glucocorticoid receptor in cytosol from human brain was studied using isoelectric focussing in slabs of polyacrylamide gel. [³H]Dexamethasone was used as tracer for receptor analysis. The glucocorticoid receptor from human brain was compared to the glucocorticoid receptor in rat brain. A similar peak of radioactivity with a pI of about 6.1 was obtained by isoelectric focussing of cytosol from both human brain and rat brain.The trypsin-induced fragmentation patterns of the glucocorticoid receptor from human brain and rat brain were very similar when analyzed by isoelectric focussing.The hormone specificity of the glucocorticoid receptor in human brain and in rat brain cytosol was compared by competition experiments using unlabelled dexamethasone, betamethasone, cortisol and corticosterone as competitors. No difference between human brain and rat brain cytosol was detected.It is concluded that the hormone specificity and the protein structure of the glucocorticoid receptors in human brain and in rat brain are similar.     

Effect of craniotomy on regional brain blood flow and ECF pH changes during isocapnic hypoxia

Regional cerebrovascular responses to 30 min of isocapnic hypoxia were examined in anesthetized rabbits. In some of the animals, a left-side craniotomy was performed and cortical ECF pH was measured either 1 mm or 2–3 mm below the surface. In the animals with open cranium, the increase in blood during hypoxia was greater in the right cortex than in the left cortex. In animals with closed cranium, the increases in blood flow were equal in both right and left crania. ECF pH measured 1 mm below the cortical surface decreased throughout the entire 30 min of exposure, while pH measured 2–3 mm below the surface was increased for the first 6 min, after which it began to decrease. Our results indicate that craniotomy reduces the local vasodilation in response to hypoxia which in turn favors a decrease in local ECF pH.     

Antioxidant effect of acetylsalicylic and salicylic acid in rat brain slices subjected to hypoxia

Acetylsalicylic acid (ASA) reduces the incidence of ischemic stroke mainly through its antithrombotic action; however, it also has a direct neuroprotective effect. The present study was designed to evaluate the effect of ASA on oxidative stress and the activity of nitric oxide synthase (NOS) in an in vitro model of hypoxia in rat brain slices. Rat brain slices were perfused with nitrogen (hypoxia) for a maximum of 120 min, after which we measured lipid peroxidation, glutathione levels, glutathione-related enzyme activities, and constitutive nitric oxide synthase (cNOS) and inducible nitric oxide synthase (iNOS) activities. In brain tissue subjected to hypoxia, ASA reduced oxidative stress and iNOS activity (all increased by hypoxia), but only when used at higher concentrations. The effects of salicylic acid (SA) were similar but more intense than were those of ASA. After oral administration, the effect of SA was much greater than that of ASA, and the decrease in cell death with SA was seen much more clearly. In view of the greater effect of SA compared to ASA on changes in oxidative stress parameters in a model of hypoxia, and higher brain concentrations of SA when it is administered alone than when ASA is given (undetectable levels), we conclude that SA plays an important role in the cytoprotective effect in brain tissue after ASA administration.     

Glutamate, glutamine and glutamine synthetase in the neonatal rat brain following hypoxia

Exposing 7-day-old rat pups to hypoxia, 8% oxygen/92% nitrogen, for 3 h alters glutamate (GLU), glutamine and glutamine synthetase (GS) activity in the striatum, frontal cortex and hippocampus. Immediately following the hypoxic insult there is a rapid transient elevation of GLU followed by a fall and then recovery to control values within 6 h. Glutamine content initially decreased after the termination of the insult, rose thereafter and approached control values within 6 h. GS activity was depressed after hypoxia and gradually returned to normal levels within 6 h. GS mRNA was increased in the three brain regions studied after hypoxia and returned to control values within 24 h. These results suggest that hypoxia alters GLU metabolism in the immature brain.     

The roles of aquaporin-4 in brain edema following neonatal hypoxia ischemia and reoxygenation in a cultured rat astrocyte model

Aquaporin-4 (AQP4), a water channel protein, is abundantly expressed in astrocytes and plays a key role in the development of brain edema. However, it is not clear whether AQP4 contributes to astrocytic swelling in hypoxia-ischemia (HI). To investigate the roles of AQP4 in astrocytic swelling during HI and reoxygenation, we measured AQP4 expression and astrocytic cellular volume in cultured rat astrocytes following HI and reoxygenation. RNA interference was used to knockdown AQP4 expression (AQP4(-/-)). Real-time polymerase chain reaction and Western blot analysis were used to detect the inhibitory efficiency of AQP4. We found that the maximal inhibition of AQP4 mRNA and protein in astrocytes after AQP4 siRNA transfection (AQP4(-/-)) was approximately 77 and 85%, respectively, compared to wild-type AQP4 (AQP4(+/+)) expression. Cellular volume in both AQP4(-/-) and AQP4(+/+) astrocytes was significantly increased during HI compared to cells cultured in normoxia (P<0.05). However, cellular volume during HI in AQP4(-/-) astrocytes was significantly less than that in AQP4(+/+) astrocytes (P<0.05). After reoxygenation, the cellular volume gradually decreased to control levels at 7 days in AQP4(-/-) but at 5 days in AQP4(+/+) astrocytes. The different roles of AQP4 during HI and reoxygenation suggest that AQP4 knockdown may protect against water influx in the formation of astrocyte swelling during HI, and may also delay water clearance in the resolution of astrocyte swelling during reoxygenation. In conclusion, AQP4 mediates bidirectional transport of water across astrocytes during HI and reoxygenation. AQP4 manipulation may serve as a novel therapeutic strategy during different periods of hypoxic-ischemic brain edema in neonates.     

Effect of torasemide on intracranial pressure, mean systemic arterial pressure and cerebral perfusion pressure in experimental brain edema of the rat

The study was performed to establish whether a lipophilic loop diuretic, torasemide could modify intracranial pressure and cytotoxic brain edema. Brain edema was induced by water intoxication in nephrectomized rats. Following intravenous injection of 100 mg torasemide/kg body weight at 50, 60, 70, 90 and 120 min, a significant decrease of intracranial pressure was observed.     

颅脑损伤大鼠肠道防御素-5 mRNA表达

目的探讨弥漫性脑损伤后肠道防御素-5（RD-5）变化。方法大鼠32只，分为对照组8只；弥漫性脑损伤组24只，再分成3个亚组（24h，3d，7d）。观察回肠RD-5mRNA的表达，回肠肠粘膜的病理改变以及血液内毒素的变化。结果弥漫性脑损伤后24hRD-5 mRNA的表达显著升高（P〈0．01），伤后3d降低至正常水平以下；回肠肠粘膜损伤，血液内毒素伤后24h最高（P〈0．01），3至7d仍显著高于正常水平（P〈0．01）。结论弥漫性脑损伤早期RD-5 mRNA的表达增强可能是机体的一种保护性反应。     

Effects of neonatal hypoxia on brain development in the rat: immediate and long-term biochemical alterations in discrete regions

To evaluate the sensitivity of immature brain tissue to hypoxic insult, neonatal rats were exposed to 7% O2 for 2 h at critical stages of development (1, 8, 15, 23 days of postnatal age); the immediate and long-term impact of hypoxia was then assessed in cerebellum, cerebral cortex and midbrain through measurement of ornithine decarboxylase (ODC) activity, a biochemical determinant of cellular injury and subsequent maturation, and through measurements of protein synthesis, growth and synaptosomal uptake of norepinephrine (an index of noradrenergic synaptogenesis). In one-day-old rats, hypoxia caused stimulation of protein synthesis and short-term suppression of ODC activity which persisted for several hours after termination of low O2 exposure; over the ensuing days, there was a prolonged elevation of enzyme activity and a subsequent, regionally selective increase in synaptosomal uptake of norepinephrine without changes in brain growth. In contrast, hypoxia in 8-day-old rats produced signs of metabolic injury, with a short-term elevation of ODC throughout the brain and reduced protein synthetic rates, eventual shortfalls in brain regional growth and no net increase in synaptosomal uptake. The effects of hypoxia on brain regional growth in 8-day-old animals appeared to represent an age-specific effect, as low as O2 conditions in older animals did not affect growth (animals made hypoxic at 15 or 23 days), but did produce an eventual reduction in synaptosomal uptake (hypoxia at 15 days). Differences between one-day-old and 8-day-old rats were also apparent in cerebral responses simply to a 2-h separation from the dam under normoxic conditions. These results support the view that cellular development and synaptogenesis are compromised when neonatal brain tissue is exposed to hypoxic conditions, and that there are critical periods of sensitivity in which processes undergoing rapid maturational change are particularly vulnerable.     

Voltage-sensitive Na channels increase in number in newborn rat brain after in utero hypoxia

The effect of chronic in utero hypoxia on voltage-sensitive Na⁺ channels in newborn rat brain was investigated by means of ligand binding and autoradiography with [³H]saxitoxin (STX,na⁺ channel ligand). We found that: (a) binding properties were different between hypoxic and control brains with alinear Scatchard plots in hypoxic brain versus linear ones in the control; (b) STX binding density increased greatly in response to hypoxic stimulation; and (c) the hypoxia-induced increase in STX binding was heteregneous in various brian areas. We conclude that hypoxia in utero alters the expression of Na⁺ channels and induces an up-regulation of Na⁺ channel density as a function of brain area. This is the first report of this phenomenon and we believe that the increased Na⁺ channels may play an important role in the etiology of neurological disorders such as epilepsy.     

Post-traumatic brain hypothermia reduces histopathological damage following concussive brain injury in the rat

The purposes of this study were (1) to document the histopathological consequences of moderate traumatic brain injury (TBI) in anesthetized Sprague-Dawley rats, and (2) to determine whether posttraumatic brain hypothermia (30°C) would protect histopathologically. Twenty-four hours prior to TBI, the fluid percussion interface was positioned over the right cerebral cortex. On the 2nd day, fasted rats were anesthetized with 70% nitrous oxide, 1% halothane, and 30% oxygen. Under controlled physiological conditions and normothermic brain temperature (37.5°C), rats were injured with a fluid percussion pulse ranging from 1.7 to 2.2 atmospheres. In one group, brain temperature was maintained at normothermic levels for 3 h after injury. In a second group, brain temperature was reduced to 30°C at 5 min post-trauma and maintained for 3 h. Three days after TBI, brains were perfusion-fixed for routine histopathological analysis. In the normothermic group, damage at the site of impact was seen in only one of nine rats. In contrast, all normothermic animals displayed necrotic neurons within ipsilateral cortical regions lateral and remote from the impact site. Intracerebral hemorrhagic contusions were present in all rats at the gray-white interface underlying the injured cortical areas. Selective neuronal necrosis was also present within the CA3 and CA4 hippocampal subsectors and thalamus. Post-traumatic brain hypothermia significantly reduced the overall sum of necrotic cortical neurons (519±122 vs 952±130, mean ±SE, P=0.03, Kruskal-Wallis test) as well as contusion volume (0.50±0.14 vs 2.14±0.71 mm³, P=0.004). These data document a consistent pattern of histopathological vulnerability following normothermic TBI and demonstrate hypothermic protection in the post-traumatic setting.Supported by USPHS Grants NS30291 and NS27127     

Effect of ischemia and hypoxia on rat brain glycolipids

Hypoxia and ischemia produced the changes in the rat brain lipid composition. Moderate decreases in the content of phospholipids, cerebrosides, and gangliosides were observed. Three different experimental models of oxygen deficiency varied as to the degree of energy disturbances were correlated with their effect on the particular lipid classes. The decrease of cerebrosides content, but not gangliosides and phospholipids, could be attributed directly to energy disturbances and concomitant restriction of glucose supply to the hypoxic-ischemic brain.     

Characterization of cellular and neurological damage following unilateral hypoxia/ischemia

Rodent models of stroke are often used to investigate the mechanisms that lead to ischemic neuronal damage. In this study, we used a model of cerebral hypoxia with ischemia to produce unilateral damage in C57Bl/6 mice. Lesion volume, ascertained by TTC staining, increased with longer durations of hypoxia. Additionally, cresyl violet, TUNEL, and FluoroJade staining showed a statistically significant increase in cellular damage in the ipsilateral cortex, CA1 pyramidal layer, and dentate gyrus of the hippocampus of ipsilateral hypoxic/ischemic tissue versus sham tissue. Astrocyte reactivity, determined by GFAP staining, was significantly higher in the ipsilateral H/I cortex and contralateral hippocampus compared to sham cortex and hippocampus, respectively. Increased microglia activation was evident in the H/I-treated cortex and hippocampus versus sham cortex and hippocampus, particularly within areas undergoing degeneration. To examine whether this model produces motor deficits, a battery of tests were administered before and after hypoxia. Following 45 min H/I, locomotor activity, rotarod performance and performance on an inverted wire hang test were all significantly decreased. These data indicate that the histological evidence of neuronal damage is consistent with functional deficits and suggest that this model may be useful for investigating strategies designed to protect neurons from hypoxia/ischemia-induced damage.     

Evaluation of neuropsychological rehabilitation following severe traumatic brain injury: A case report

This case study presents the evaluation of traumatic brain injury rehabilitation for Jane, a 35-year-old woman who sustained a severe traumatic brain injury (TBI). On-going rehabilitation addressed difficulties within a structured neuro-rehabilitative therapeutic milieu and was holistically evaluated. Results showed improvement in mood and quality of life and at the compensatory level for cognition. At the impairment level, many of the affected areas of cognition remained at least below predicted pre-morbid levels. This supports the view that although TBI impairments may be permanent, structured neuro-rehabilitation can be effective through external and compensatory aids. Recording this holistically contributes to evidencing this.     

Chromatographic and immunochemical characterization of rat brain neuropeptide Y-like immunoreactivity (NPY-LI) following repeated electroconvulsive,stimuli

The present work investigates the possible existence of molecular heterogeneity of endogenous neuropeptide Y (NPY) in the rat brain and, if existing, whether it is affected by repeated electroconvulsive stimuli (ECS). Different column chromatographic techniques (gel-permeation chromatography, HPLC, ion-pair reverse-phase HPLC) were used combined with immunochemical methods based on antisera directed towards two different epitopes of NPY (antiserum N1, directed towards the midportion of NPY, and antiserum C1, directed towards the C-terminal end of NPY). Following repeated ECS increased concentrations of NPY were seen in the occipital cortex and hippocampus (P<0.001) when analyzed by direct radioimmunoassays (RIA). Chromatographic characterization showed that the NPY mainly consisted of intact NPY (1-36) and sulphoxidated form of NPY, no short C-terminal homologues were found. This is of importance since it has been shown that NPY (1-36) has different biological properties compared to C-terminal homologues. © 1995 Wiley-Liss, Inc.     

Effects of ischemia-hypoxia induced by interruption of uterine blood flow on fetal rat liver and brain enzyme activities and offspring behavior

The effects of acute perinatal ischemia-hypoxia on fetal liver and brain energy metabolism, fetal brain total free fatty acid concentration and subsequent offspring behavior were investigated in rats. Ischemia-hypoxia was induced at term either by ligation of the uterine blood vessels or submersion of the entire uterine horn in warmed saline. Fetuses of the adjacent horn served as within-dam controls for all assessments and fetuses of dams which had not undergone the surgical stress served as independent controls for enzyme assays. Ischemia-hypoxia was associated with reduced activity of fatty acid synthase in the liver and brain. Total free fatty acid concentration significantly increased in the fetal hypoxic brain. Pups not used for enzyme analyses were cross-fostered for behavioral assessments. Relative to the enzymatic alterations, there were few behavioral alterations associated with ischemia-hypoxia. At postnatal day 30, rats made hypoxic by ligation of the uterine blood vessels had decreased caudate nucleus and brain stem weights relative to within-dam controls. At postnatal day 85, rats made hypoxic by submersion of the uterine horn had decreased olfactory bulb weight. The results of this study indicate an initial acute response to a brief period of ischemia-hypoxia at term pregnancy in the fetal rat brain and liver.     

Early postnatal development of rat brain: in vivo diffusion tensor imaging

Perinatal hypoxia is a major cause of neurodevelopmental deficits. Neuronal migration patterns are particularly sensitive to perinatal hypoxia/ischemia and are associated with the clinical deficits. The rat model of hypoxia/ischemia at P7 mimics that of perinatal injury in humans. Before assessing the effects of postnatal injury on brain development, it is essential to determine the normal developmental trajectories of various brain structures in individual animals. In vivo longitudinal diffusion tensor imaging (DTI) was performed from postnatal day 0 (P0) to P56 on Wistar rats. The DTI metrics, mean diffusivity (MD), fractional anisotropy (FA), axial (lambdal) and radial (lambdat) diffusivities, were determined for four gray matter and eight white matter structures. The FA of the cortical plate and the body of corpus callosum decreased significantly during the first 3 weeks after birth. The decrease in the cortical plate's FA value was associated mainly with an increase in lambdat. The initial decrease in FA of corpus callosum was associated with a significant decrease in lambdal. The FA of corpus callosum increased during the rest of the observational period, which was mainly associated with a decrease in lambdat. The FA of gray matter structures, hippocampus, caudate putamen, and cortical mantle did not show significant changes between P0 and P56. In contrast, the majority of white matter structures showed significant changes between P0 and P56. These temporal changes in the DTI metrics were related to the neuronal and axonal pruning and myelination that are known to occur in the developing brain.