Effect of long-term (2 years) exposure of mouse brains to global system for mobile communication (GSM) radiofrequency fields on astrocytic immunoreactivity.

This study was designed to determine whether long-term (2 years) brain exposure to mobile telephone radiofrequency (RF) fields produces any astrocytic activation as these glia react to a wide range of neural perturbations by astrogliosis. Using a purpose-designed exposure system at 900 MHz, mice were given a single, far-field whole body exposure at a specific absorption rate of 4 W/kg on five successive days per week for 104 weeks. Control mice were sham-exposed or freely mobile in a cage to control any stress caused by immobilization in the exposure module. Brains were perfusion-fixed with 4% paraformaldehyde and three coronal levels immunostained for glial fibrillary acidic protein (GFAP). These brain slices were then examined by light microscopy and the amount of this immunomarker quantified using a color deconvolution method. There was no change in astrocytic GFAP immunostaining in brains after long-term exposure to mobile telephony microwaves compared to control (sham-exposed or freely moving caged mice). It was concluded that long-term (2 years) exposure of murine brains to mobile telephone RF fields did not produce any astrocytic reaction (astrogliosis) detectable by GFAP immunostaining.

Substance P antagonists as a novel intervention for brain edema and raised intracranial pressure.

Increased intracranial pressure (ICP) following acute brain injury requires the accumulation of additional water in the intracranial vault. One source of such water is the vasculature, although the mechanisms associated with control of blood-brain barrier permeability are unclear. We have recently shown that acute brain injury, such as neurotrauma and stroke, results in perivascular accumulation of the neuropeptide, substance P. This accumulation is associated with increased blood-brain barrier permeability and formation of vasogenic edema. Administration of a substance P antagonist targeting the tachykinin NK1 receptor profoundly reduced the increased blood-brain barrier permeability and edema formation, and in small animal models of acute brain injury, improved functional outcome. In a large, ovine model of experimental traumatic brain injury, trauma resulted in a significant increase in ICP. Administration of an NK1 antagonist caused a profound reduction in post--traumatic ICP, with levels returning to normal within 4 h of drug administration. Substance P NK1 antagonists offer a novel therapeutic approach to the treatment of acute brain injury.

Alterations in membrane potential in mitochondria isolated from brain subregions during focal cerebral ischemia and early reperfusion: evaluation using flow cytometry.

Mitochondria isolated from brain tissue following middle cerebral artery occlusion or during early reperfusion were tested for their ability to generate a membrane potential under standard conditions in vitro. Membrane potential was evaluated based on rhodamine 123 fluorescence in the mitochondria as detected using flow cytometry. Compared with equivalent samples from the contralateral hemisphere, the geometric mean fluorescence was significantly lower in mitochondria prepared from the striatum and perifocal tissue in the cortex at 3 h ischemia. During reperfusion, this property was decreased in mitochondria from tissue in the striatum and cortex that had been part of severely ischemic core tissue during the arterial occlusion. These findings provide additional evidence that mitochondria develop changes during ischemia and reperfusion that are likely to limit their ability to respond to changing energy requirements and contribute to cell dysfunction and cell death. It also demonstrates the ability to gain a sensitive measure of these mitochondrial changes using flow cytometry.

Astrocytic function assessed from 1-14C-acetate metabolism after temporary focal cerebral ischemia in rats.

Astrocytes play many roles essential for normal brain activity. The ability of these cells to recover after temporary focal cerebral ischemia is likely to be one important determinant of the extent of brain dysfunction and tissue damage. We have assessed astrocytic function based on the incorporation of radiolabel from 1-14C-acetate into glutamine at 1 hour of recirculation after middle cerebral artery occlusion for 2 or 3 hours in rats. There were marked differences in the response between subregions within the tissue subjected to ischemia, but the overall pattern of changes was similar after each ischemic period. The striatum, which forms part of the severely ischemic focal tissue during arterial occlusion, showed a large (44% to 68%) decrease in glutamine labeling compared with equivalent tissue from the contralateral hemisphere. In contrast, 14C-glutamine content was not significantly altered in perifocal tissue in the cerebral cortex, which was subjected to more moderate ischemia. Cortical focal tissue also was not significantly affected, but the response was much more variable between rats. In these brain subregions, the extent of recovery of the 14C-acetate metabolism after ischemia was not a good predictor of the likelihood of subsequent infarct development. Interestingly, a similar pattern of responses persisted when recirculation was extended to 4 hours. These results indicate that many astrocytes, particularly in the cortex, remain viable and capable of at least some complex oxidative metabolism during the first few hours of recirculation.

(-)-Epigallocatechin gallate as an intervention for the acute treatment of cerebral ischemia.

This study examined the neuroprotective effects and possible hepatotoxicity of (-)-epigallocatechin gallate (EGCG) in a rat model of transient focal cerebral ischemia. Male Sprague-Dawley rats (265-295 g) were treated with either 50 mg kg(-1) of EGCG or saline, i.p., immediately post-ischemia and every day thereafter, in a middle cerebral artery occlusion model of stroke. Sacrifice occurred 72 h post-ischemia and 2,3,5-triphenyltetrazolium chloride staining was used to quantify neuronal infarction. Hepatotoxicity was determined by taking blood samples for plasma alanine aminotransferase (ALT) activity. Spleen, kidney, liver and testes wet weights were also recorded. Total infarct volume was significantly (P<0.05) reduced in the EGCG-treated group as compared to controls. Analysis of the mean infarct area showed a significant (P<0.05) decrease in slices 6 and 7 in the EGCG-treated group. No significant differences were found in organ weights or ALT levels between treatment groups. Our findings, in part, validate and extend previous observations illustrating that 50 mg kg(-1), i.p. EGCG is non-toxic and neuroprotective. However, we also found that EGCG treatment appreciably increased (>50%) the number of animals that developed an intracerebral hemorrhage. We therefore conclude that 50 mg kg(-1) EGCG is not a viable intervention for the acute treatment of cerebral ischemia, as it is likely to increase the risk of intracerebral hemorrhaging.

Elevated Intracranial Pressure and Cerebral Edema following Permanent MCA Occlusion in an Ovine Model.

INTRODUCTION: Malignant middle cerebral artery (MCA) stroke has a disproportionately high mortality due to the rapid development of refractory space-occupying cerebral edema. Animal models are essential in developing successful anti-edema therapies; however to date poor clinical translation has been associated with the predominately used rodent models. As such, large animal gyrencephalic models of stroke are urgently needed. The aim of the study was to characterize the intracranial pressure (ICP) response to MCA occlusion in our recently developed ovine stroke model. MATERIALS AND METHODS: 30 adult female Merino sheep (n = 8-12/gp) were randomized to sham surgery, temporary or permanent proximal MCA occlusion. ICP and brain tissue oxygen were monitored for 24 hours under general anesthesia. MRI, infarct volume with triphenyltetrazolium chloride (TTC) staining and histology were performed. RESULTS: No increase in ICP, radiological evidence of ischemia within the MCA territory but without space-occupying edema, and TTC infarct volumes of 7.9+/-5.1% were seen with temporary MCAO. Permanent MCAO resulted in significantly elevated ICP, accompanied by 30% mortality, radiological evidence of space-occupying cerebral edema and TTC infarct volumes of 27.4+/-6.4%. CONCLUSIONS: Permanent proximal MCAO in the sheep results in space-occupying cerebral edema, raised ICP and mortality similar to human malignant MCA stroke. This animal model may prove useful for pre-clinical testing of anti-edema therapies that have shown promise in rodent studies.

Losses of NG2 and NeuN immunoreactivity but not astrocytic markers during early reperfusion following severe focal cerebral ischemia.

The ability of glia to recover essential functions following a period of focal cerebral ischemia is likely to be one important factor influencing the severity of tissue damage that subsequently develops. In this study, we have compared changes in immunoreactivity of markers specific for astrocytes, NG2-positive glia and neurons in tissue subregions during early reperfusion following 3 h of middle cerebral artery occlusion to provide insights into possible differential susceptibility of these cell populations. Under the conditions used, infarction ultimately encompasses most of the perfusion territory of the occluded artery. Nonetheless, alterations in immunoreactivity during the first 3 h of recirculation were restricted to brain regions that had been subjected to severe ischemia. In the striatum, cellular immunoreactivity for NG2 and neuronal markers, NeuN and microtubule-associated protein 2, was greatly reduced by 1 h of reperfusion and declined further at 3 h. NG2 labeling of blood vessels in the striatum appeared post-ischemically, mimicking expression of this protein during development. Less severe changes were seen in the neuronal markers in overlying cerebral cortex. In contrast to the losses of other cellular proteins, immunoreactivity for the astrocytic marker, glial fibrillary acidic protein, was preserved in all tissue that had been subjected to severe ischemia and labeling of another astrocytic protein, glutamine synthetase, was increased by 3 h of reperfusion. These findings provide the first evidence of marked sensitivity of NG2-immunoreactivity to severe ischemia and suggest a greater initial resistance of astrocytes compared with neurons and NG2-positive glia to ischemia-reperfusion damage.

Automatic nonsubjective estimation of antigen content visualized by immunohistochemistry using color deconvolution.

We describe a method for the automatic, nonsubjective estimation of 3,3' diaminobenzidine (DAB) in digital images obtained from routine central nervous system immunohistochemistry using freely available, platform-independent public domain image processing software. This technique estimates the amount of antigen visualized but does not measure antigen content directly. Combined with whole brain section high-resolution scanning, a "virtual dissection" (extracting the region of interest) makes it possible to estimate relative antigen content in either subcellular structures, specific brain regions, or in whole tissue sections at magnifications up to 40×. The digital image is processed using Ruifrok and Johnston's color deconvolution method to separate the brown DAB chromogen from the hematoxylin counterstain on a microscope slide. A monochrome image representing the DAB content is then subjected to frequency analysis using NIH-ImageJ and a weighting calculation to estimate the amount of DAB (antigen) as a dimensionless index. The method described produces results that agree with enzyme-linked immunosorbent assays, and is automatic and nonsubjective. The method could easily be adapted to other types of tissue or cell cultures.

Further investigations into the speed of cerebral swelling following blunt cranial trauma.

An anesthetized sheep model of traumatic brain injury (TBI) has been developed to assess early changes in intracranial pressure (ICP) following closed head injury. Immediately after TBI, a transient (<10 min) hypertensive response occurred, followed by significant and prolonged systemic hypotension. ICP demonstrated a biphasic response, being seven times baseline values of 8 ± 2 mm Hg 10 min after injury, decreasing to 25 ± 2 mm Hg by 30 min, and then increasing to values exceeding 30 mm Hg by 4 h postinjury. ICP was always significantly higher than baseline values, which combined with hypotension, reduced cerebral perfusion pressure to less than 60% of normal. This early and sustained increase in ICP after craniocerebral trauma acutely alters cerebral perfusion pressure and brain oxygenation and provides a potential pathophysiological explanation for immediate clinical manifestations in humans following significant TBI.

A surgical model of permanent and transient middle cerebral artery stroke in the sheep.

BACKGROUND: Animal models are essential to study the pathophysiological changes associated with focal occlusive stroke and to investigate novel therapies. Currently used rodent models have yielded little clinical success, however large animal models may provide a more suitable alternative to improve clinical translation. We sought to develop a model of acute proximal middle cerebral artery (MCA) ischemic stroke in sheep, including both permanent occlusion and transient occlusion with reperfusion. MATERIALS AND METHODS: 18 adult male and female Merino sheep were randomly allocated to one of three groups (n = 6/gp): 1) sham surgery; 2) permanent proximal MCA occlusion (MCAO); or 3) temporary MCAO with aneurysm clip. All animals had invasive arterial blood pressure, intracranial pressure and brain tissue oxygen monitoring. At 4 h following vessel occlusion or sham surgery animals were killed by perfusion fixation. Brains were processed for histopathological examination and infarct area determination. 6 further animals were randomized to either permanent (n = 3) or temporary MCAO (n = 3) and then had magnetic resonance imaging (MRI) at 4 h after MCAO. RESULTS: Evidence of ischemic injury in an MCA distribution was seen in all stroke animals. The ischemic lesion area was significantly larger after permanent (28.8%) compared with temporary MCAO (14.6%). Sham animals demonstrated no evidence of ischemic injury. There was a significant reduction in brain tissue oxygen partial pressure after permanent vessel occlusion between 30 and 210 mins after MCAO. MRI at 4 h demonstrated complete proximal MCA occlusion in the permanent MCAO animals with a diffusion deficit involving the whole right MCA territory, whereas temporary MCAO animals demonstrated MRA evidence of flow within the right MCA and smaller predominantly cortical diffusion deficits. CONCLUSIONS: Proximal MCAO can be achieved in an ovine model of stroke via a surgical approach. Permanent occlusion creates larger infarct volumes, however aneurysm clip application allows for reperfusion.

Human adult dental pulp stem cells enhance poststroke functional recovery through non-neural replacement mechanisms.

Human adult dental pulp stem cells (DPSCs), derived from third molar teeth, are multipotent and have the capacity to differentiate into neurons under inductive conditions both in vitro and following transplantation into the avian embryo. In this study, we demonstrate that the intracerebral transplantation of human DPSCs 24 hours following focal cerebral ischemia in a rodent model resulted in significant improvement in forelimb sensorimotor function at 4 weeks post-treatment. At this time, 2.3 ± 0.7% of engrafted cells had survived in the poststroke brain and demonstrated targeted migration toward the stroke lesion. In the peri-infarct striatum, transplanted DPSCs differentiated into astrocytes in preference to neurons. Our data suggest that the dominant mechanism of action underlying DPSC treatment that resulted in enhanced functional recovery is unlikely to be due to neural replacement. Functional improvement is more likely to be mediated through DPSC-dependent paracrine effects. This study provides preclinical evidence for the future use of human DPSCs in cell therapy to improve outcome in stroke patients.

A substance P antagonist improves outcome when administered 4 h after onset of ischaemic stroke.

Previous studies have suggested that substance P (SP) plays a critical role in the development of brain oedema and functional deficits following traumatic brain injury and that SP receptor antagonism may improve outcome. No studies have described such a role in ischemic stroke. The present study characterized the effects of the NK1 tachykinin receptor antagonist, n-acetyl-L-tryptophan (NAT), on blood-brain barrier (BBB) breakdown, oedema formation, infarct volume and functional outcome following reversible ischemic stroke in rats. Ischemia was induced using a reversible thread model of middle cerebral artery occlusion where occlusion was maintained for 2 h before reperfusion. Animals received either NAT or equal volume saline vehicle intravenously at 2 h post-reperfusion. Ischaemic stroke resulted in increased perivascular SP immunoreactivity at 24 h. Administration of NAT significantly reduced oedema formation and BBB permeability at 24 h post-ischemia and significantly improved functional outcome as assessed over 7 days. There was no effect on infarct volume. We conclude that inhibition of SP activity with a NK1 tachykinin receptor antagonist is effective in reducing cerebral oedema, BBB permeability and functional deficits following reversible ischemia and may therefore represent a novel therapeutic approach to the treatment of ischaemic stroke.

Characterizing the role of the neuropeptide substance P in experimental subarachnoid hemorrhage.

BACKGROUND: Raised intracranial pressure (ICP) following SAH predicts poor outcome and is due to hemorrhage volume and possibly, brain edema, hydrocephalus and increased volume of circulating intracranial blood. Interventions that reduce edema may therefore reduce ICP and improve outcome. The neuropeptide substance P (SP) mediates vasogenic edema formation in animal models of ischemic stroke, intracerebral hemorrhage and brain trauma, and may contribute to development of increased ICP. SP (NK1 tachykinin receptor) blockade using n-acetyl-l-tryptophan (NAT) reduces edema and improves outcome in these models. This study therefore assessed whether SP mediates edema formation in experimental SAH. METHODS: SAH was induced in rats by either injection of autologous blood into the prechiasmatic cistern (injection SAH) or by arterial puncture of the Circle of Willis (filament SAH). NAT was injected (i.v.) 30min after SAH induction. Subgroups were assessed for brain water content, SP and albumin immunoreactivity, and functional outcome at 5, 24 and 48h or ICP over 5h. RESULTS: A secondary ICP increase occurred within 2h of SAH. Brain edema followed filament SAH (p<0.001) and correlated with functional deficits (r=0.8, p<0.01). Increased albumin immunoreactivity (p<0.001) indicated vasogenic edema. However, NAT treatment did not improve ICP, edema or outcome. CONCLUSIONS: Experimental SAH produced secondary ICP elevation, vasogenic brain edema and functional deficits, although it is unclear if edema contributed to ICP. Blockade of SP did not improve any outcome parameters, suggesting that neurogenic inflammation may be less critical than other factors in these models.

Intracranial pressure changes following traumatic brain injury in rats: lack of significant change in the absence of mass lesions or hypoxia.

Traumatic brain injury (TBI) often causes raised intracranial pressure (ICP), with >50% of all TBI- related deaths being associated with this increase in ICP. To date, there is no effective pharmacological treatment for TBI, partly because widely used animal models of TBI may not replicate many of the pathophysiological responses observed in humans, and particularly the ICP response. Generally, rodents are the animal of choice in neurotrauma research, and edema formation has been demonstrated in rat models; however, few studies in rats have specifically explored the effects of TBI on ICP. The aim of the current study was to investigate the ICP response of rats in two different, focal and diffuse, injury models of TBI. Adult male Sprague-Dawley rats were subjected to brain trauma by either lateral fluid percussion or impact-acceleration induced injury, in the presence or absence of secondary hypoxia. ICP, mean arterial blood pressure (MABP), and cerebral perfusion pressure (CPP) were monitored for 4 h after TBI. TBI alone or coupled with hypoxia did not result in any significant increase of ICP in rats unless there was an intracranial hemorrhage. At all other times, changes in CPP were the result of changes in MABP and not ICP. Our results suggest that rats may be able to compensate for the intracranial expansion associated with cerebral edema after TBI, and that they only develop a consistent post-traumatic increase in ICP in the presence of a mass lesion. Therefore, they are an inappropriate model for the investigation of ICP changes after TBI, and for the development of therapies targeting ICP.

Transient receptor potential melastatin 2 expression is increased following experimental traumatic brain injury in rats.

Traumatic brain injury (TBI) elicits a sequence of complex biochemical changes including oxidative stress, oedema, inflammation and excitotoxicity. These factors contribute to the high morbidity and mortality following TBI, although their underlying molecular mechanisms remain poorly understood. Transient receptor potential melastatin 2 (TRPM2) is a non-selective cation channel, highly expressed in the brain and immune cells. Recent studies have implicated TRPM2 channels in processes involving oxidative stress, inflammation and cell death. However, no studies have investigated the role of TRPM2 in TBI pathophysiology. In the present study, we have characterised TRPM2 mRNA and protein expression following experimental TBI. Adult male Sprague Dawley rats were injured using the impact-acceleration model of diffuse TBI with survival times between 5 and 5 days. Real-time RT-PCR (including reference gene validation studies) and semi-quantitative immunohistochemistry were used to quantify TRPM2 mRNA and protein levels, respectively, following TBI. Significant increases in TRPM2 mRNA and protein expression were observed in the cerebral cortex and hippocampus of injured animals, suggesting that TRPM2 may contribute to TBI injury processes such as oxidative stress, inflammation and neuronal death. Further characterisation of how TRPM2 may contribute to TBI pathophysiology is warranted.

Hemoglobin crystals: a pro-inflammatory potential confounder of rat experimental intracerebral hemorrhage.

In vivo rat hemoglobin crystallization has been reported in lung, liver and kidney, but never following central nervous system injury. In the present study, we examined hemoglobin crystallization following experimental intracerebral hemorrhage (ICH) and its effects on inflammation. Ninety-one rat brains, subjected to either autologous or collagenase ICH, and vehicle controls, were retrospectively examined. In both models, hemoglobin crystals were present in most brains at 24 and 48 h. They were especially prominent at 24 h in autologous ICH brains (2.5% of the hematoma vs 0.6% in collagenase animals; p=0.0001) and, at 5 h, were only present in autologous ICH brains. Crystals were diminishing at 48 h and were absent at 7 days. Crystals appeared in clusters around blood vessels. In both models, at 24 h, crystals appeared strongly chemotactic for neutrophils. This effect was most pronounced in autologous ICH brains (2628+/-182 neutrophils/mm(2) hematoma crystals vs 327+/-54 neutrophils/mm(2) hematoma; p<0.0001). In these animals up to 30% of the total neutrophilic infiltrate was located around crystals. A greater overall neutrophilic infiltrate was seen in autologous ICHs with higher percentages of crystalline hemoglobin (p=0.04 for trend). Although hemoglobin crystallization occurs in both models of ICH, it is particularly prominent following autologous ICH. Accordingly, hemoglobin crystallization may exaggerate the importance of inflammation in this model.

Inhibition of nitric oxide synthase with 7-nitroindazole does not modify early metabolic recovery following focal cerebral ischemia in rats.

Nitric oxide has been strongly implicated in the development of tissue infarction in response to focal cerebral ischemia. Nitric oxide and its derivatives can inhibit components of the electron transport chain, providing a likely target for these substances in ischemic and post-ischemic brain. Lactate content is increased during post-ischemic reperfusion in tissue destined to become infarcted, consistent with impairment of mitochondrial respiration. To investigate the possible involvement of nitric oxide in generating these changes, we have tested the effect of 7-nitroindazole, a nitric oxide synthase (NOS) inhibitor, on the content of lactate and other metabolites during early reperfusion following temporary focal ischemia. This treatment inhibited total NOS by approximately 50%. However, the treatment did not significantly affect the marked increases in lactate in post-ischemic brain nor did it alter the recovery of other energy-related metabolites. These findings indicate that inhibition of oxidative metabolism is probably not the primary site of the deleterious effects of nitric oxide and derivatives during early post-ischemic reperfusion.

The metabolism of C-glucose by neurons and astrocytes in brain subregions following focal cerebral ischemia in rats.

To provide insights into the effects of temporary focal ischemia on the function of neurons and astrocytes in vivo, we measured the incorporation of radiolabel from [U-14C]glucose into both glutamate and glutamine in brain subregions at 1 h of reperfusion following occlusion of the middle cerebral artery for 2 or 3 h. Under the experimental conditions used, 14C-glutamate is mainly produced in neurons whereas 14C-glutamine is generated in astrocytes from 14C-glutamate of both neuronal and astrocytic origin. Radiolabel incorporation into both amino acids was greatly decreased. The change in 14C-glutamate accumulation provides strong evidence for substantial reductions in neuronal glucose metabolism. The resulting decrease in delivery of 14C-glutamate from the neurons to astrocytes was probably also the major contributor to the change in 14C-glutamine content. These alterations probably result in part from a marked depression of glycolytic activity in the neurons, as suggested by previous studies assessing deoxyglucose utilization. Alterations in 14C-glucose metabolism were not restricted to tissue that would subsequently become infarcted. Thus, these changes did not inevitably lead to death of the affected cells. The ATP : ADP ratio and phosphocreatine content were essentially preserved during recirculation following 2 h of ischemia and showed at most only moderate losses in some subregions following 3 h of ischemia. This retention of energy reserves despite the decreases in 14C-glucose metabolism in neurons suggests that energy needs were substantially reduced in the post-ischemic brain. Marked increases in tissue lactate accumulation during recirculation, particularly following 3 h of ischemia, provided evidence that impaired pyruvate oxidation probably also contributed to the altered 14C-glucose metabolism. These findings indicate the presence of complex changes in energy metabolism that are likely to greatly influence the responses of neurons and astrocytes to temporary focal ischemia.