Stephen C. Helps

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.

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 Johnstons 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.

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.

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.

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.

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.

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.

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.

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.