James P. McAllister

Exercise preconditioning ameliorates inflammatory injury in ischemic rats during reperfusion

There is evidence that physical activity is associated with decreased brain injury resulting from transient middle cerebral artery (MCA) occlusion. We investigated whether exercise could reduce stroke-induced brain inflammatory injury and its associated mediators. Sprague Dawley rats (3xa0months old) were subjected to 30xa0min exercise on a treadmill each day for 1–3xa0weeks. Stroke, in exercised and non-exercised animals, was then induced by a 2-h MCA occlusion followed by 48xa0h of reperfusion using an intraluminal filament. Endothelial expression of the intercellular adhesion molecule 1 (ICAM-1) and leukocyte infiltration were determined by immunocytochemistry. Expressions of tumor necrosis factor-α (TNF-α) and ICAM-1 mRNA were detected using a real-time reverse transcriptase-polymerase chain reaction in ischemic rats with or without exercise, and in non-ischemic control rats following exercise. Expression of TNF-α increased after exercise for 2 and 3xa0weeks. The overexpression of TNF-α was not further elevated in 3-week exercised rats subjected to a transient MCA occlusion and 6 or 12xa0h of reperfusion, as compared to that in non-exercised rats. Furthermore, ICAM-1 mRNA expression remained at significantly (P<0.01) low levels in exercised animals during ischemia/reperfusion. Pre-ischemic exercise significantly (P<0.01) reduced numbers of ICAM-1-positive vessels and infiltrating leukocytes in the frontoparietal cortex and dorsolateral striatum in ischemic rats after 48xa0h of reperfusion. Exercised ischemic rats demonstrated an 11±7% infarct volume of contralateral hemisphere as compared to a 52±3% volume in non-exercised ischemic rats. The data suggests that exercise inhibits inflammatory injury (i.e., decreased expression of inflammatory mediators and reduced accumulation of leukocytes) during reperfusion, leading to reduced brain damage. Chronically increased expression of TNF-α during exercise prevent the same downstream inflammatory events as does acutely elevated TNF-α after ischemia/reperfusion.

Increased astrocyte proliferation in rats after running exercise

The aim in this study was to investigate whether physical exercise could induce astroglial proliferation in the frontoparietal cortex and dorsolateral striatum where extensive angiogenesis had been found after exercise in previous studies. Adult male Sprague Dawley rats (n=48) were used in four experimental groups. Animals were exercised 30 min each day on a treadmill on which repetitive locomotor movement was required, for 0 (n=12), 3 (n=12) or 6 (n=12) weeks, as well as 3-week exercise plus 3-week rest (n=12). Brain tissues of the exercised and non-exercised rats were processed for glial fibrillary acidic protein (GFAP) immunocytochemistry (n=6 x 4) and Western blotting (n=6 x 4) to evaluate regional astrocyte proliferation in the frontoparietal cortex and dorsolateral striatum. By using GFAP immunocytochemistry and stereological methods, we compared the density of astrocytes in the animals with or without exercise. In comparison to non-exercised animals, a significant (p<0.01) increase in the number of astrocytes was observed in both cortex and striatum of rats exercised for 3 or 6 weeks. Our data also indicated that astrocytic density continued to increase up to 6 weeks either with an additional 3 weeks of exercise (p<0.01) or 3 weeks of rest (p<0.01). In addition, Western blotting analysis showed an obvious increase in GFAP protein from cortex and striatum of exercised animals. Astrocytosis after exercise, coupled with angiogenesis, is thought to provide strength to the neurovascular unit (a construct consisting of microvascular endothelium, astroglia, neurons and the extracellular matrix). Strengthening of this unit by exercise may protect blood-brain-barrier function following brain injury, such as that occurring after stroke.

Influence of silicone surface roughness and hydrophobicity on adhesion and colonization of Staphylococcus epidermidis

Bacterial adhesion and colonization are complicated processes that depend on many factors, including surface chemistry, hydrophobicity, and surface roughness. The contribution of each of these factors has not been fully elucidated because most previous studies used different polymeric surfaces to achieve differences in properties. The objective of this study was to modify hydrophobicity and roughness on one polymeric surface, eliminating the confounding contribution of surface chemistry. Mechanically assembled monolayer (MAM) preparation methods (both one- and two-dimensional) were used to impart different degrees of hydrophobicity on fluoroalkylsilane (FAS)-coated silicone. Surface roughness was varied by casting the silicone to templates prepared with different abrasives. Surface hydrophobicity was determined by contact angle measurement, whereas surface roughness was determined by scanning electron microscopy (SEM) and atomic force microscopy (AFM). Bacterial adhesion and colonization were analyzed using a direct colony-counting method and SEM images. Hydrophobicity increased as a function of stretched length or width (Deltax or Deltay); it reached a maximum at Deltax = 60% with one-dimensional MAM and decreased as Deltax further increased to 80 and 100%. The same trend was observed for the two-dimensional MAM. After 12-h incubation, all the FAS/silicone surfaces had significantly reduced adherence of Staphylococcus epidermidis by 42-89%, compared to untreated silicone, and the degree of which is inversely related to surface hydrophobicity. On the other hand, surface roughness had a significant effect on bacterial adhesion and colonization only when the root-mean-square roughness was higher than 200 nm.

Reduction of astrogliosis and microgliosis by cerebrospinal fluid shunting in experimental hydrocephalus.

BackgroundReactive gliosis has the potential to alter biomechanical properties of the brain, impede neuronal regeneration and affect plasticity. Determining the onset and progression of reactive astrogliosis and microgliosis due to hydrocephalus is important for designing better clinical treatments.MethodsReactive astrogliosis and microgliosis were evaluated as the severity of hydrocephalus increased with age in hydrocephalic H-Tx rats and control littermates. Previous studies have suggested that gliosis may persist after short-term drainage (shunt treatment) of the cerebrospinal fluid. Therefore shunts were placed in 15d hydrocephalic rats that were sacrificed after 6d (21d of age) or after 21d (36d of age). Tissue was processed for Western blot procedures and immunohistochemistry, and probed for the astrocytic protein, Glial Fibrillary Acidic Protein (GFAP) and for microglial protein, Isolectin B4 (ILB4).ResultsIn the parietal cortex of untreated hydrocephalic animals, GFAP levels increased significantly at 5d and at 12d compared to age-matched control rats. There was a continued increase in GFAP levels over control at 21d and at 36d. Shunting prevented some of the increase in GFAP levels in the parietal cortex. In the occipital cortex of untreated hydrocephalic animals, there was a significant increase over control in levels of GFAP at 5d. This trend continued in the 12d animals, although not significantly. Significant increases in GFAP levels were present in 21d and in 36d animals. Shunting significantly reduced GFAP levels in the 36d shunted group. Quantitative grading of immuno-stained sections showed similar changes in GFAP stained astrocytes.Immuno-stained microglia were altered in shape in hydrocephalic animals. At 5d and 12d, they appeared to be developmentally delayed with a lack of processes. Older 21d and 36d hydrocephalic animals exhibited the characteristics of activated microglia, with thicker processes and enlarged cell bodies. Following shunting, fewer activated microglia were present.Histologic examination of the periventricular area and the periaqueductal area showed similar findings with the 21d and 36d animals having increased populations of both astrocytes and microglia which were reduced following shunting with a more dramatic reduction in the long term shunted animals.ConclusionOverall, these results suggest that reactive astrocytosis and microgliosis are associated with progressive untreated ventriculomegaly, but that shunt treatment can reduce the gliosis occurring with hydrocephalus.

Priorities for hydrocephalus research: report from a National Institutes of Health–sponsored workshop

OBJECTnTreatment for hydrocephalus has not advanced appreciably since the advent of cerebrospinal fluid (CSF) shunts more than 50 years ago. Many questions remain that clinical and basic research could address, which in turn could improve therapeutic options. To clarify the main issues facing hydrocephalus research and to identify critical advances necessary to improve outcomes for patients with hydrocephalus, the National Institutes of Health (NIH) sponsored a workshop titled Hydrocephalus: Myths, New Facts, and Clear Directions. The purpose of this paper is to report on the recommendations that resulted from that workshop.nnnMETHODSnThe workshop convened from September 29 to October 1, 2005, in Bethesda, Maryland. Among the 150 attendees was an international group of participants, including experts in pediatric and adult hydrocephalus as well as scientists working in related fields, neurosurgeons, laboratory-based neuroscientists, neurologists, patient advocates, individuals with hydrocephalus, parents, and NIH program and intramural staff. Plenary and breakout sessions covered injury and recovery mechanisms, modeling, biomechanics, diagnosis, current treatment and outcomes, complications, quality of life, future treatments, medical devices, development of research networks and information sharing, and education and career development.nnnRESULTSnThe conclusions were as follows: 1) current methods of diagnosis, treatment, and outcomes monitoring need improvement; 2) frequent complications, poor rate of shunt survival, and poor quality of life for patients lead to unsatisfactory outcomes; 3) investigators and caregivers need additional methods to monitor neurocognitive function and control of CSF variables such as pressure, flow, or pulsatility; 4) research warrants novel interdisciplinary approaches; 5) understanding of the pathophysiological and recovery mechanisms of neuronal function in hydrocephalus is poor, warranting further investigation; and 6) both basic and clinical aspects warrant expanded and innovative training programs.nnnCONCLUSIONSnThe research priorities of this workshop provide critical guidance for future research in hydrocephalus, which should result in advances in knowledge, and ultimately in the treatment for this important disorder and improved outcomes in patients of all ages.

Local saline infusion into ischemic territory induces regional brain cooling and neuroprotection in rats with transient middle cerebral artery occlusion.

OBJECTIVEThe neuroprotective effect of hypothermia has long been recognized. Use of hypothermia for stroke therapy, which is currently being induced by whole-body surface cooling, has been limited primarily because of management problems and severe side effects (e.g., pneumonia). The goal of this study was to determine whether local infusion of saline into ischemic territory could induce regional brain cooling and neuroprotection. METHODSA novel procedure was used to block the middle cerebral artery of rats for 3 hours with a hollow filament and locally infuse the middle cerebral artery-supplied territory with 6 ml cold saline (20°C) for 10 minutes before reperfusion. RESULTSThe cold saline infusion rapidly and significantly reduced temperature in cerebral cortex from 37.2 ± 0.1 to 33.4 ± 0.4°C and in striatum from 37.5 ± 0.2 to 33.9 ± 0.4°C. The significant hypothermia remained for up to 60 minutes after reperfusion. Significant (P < 0.01) reductions in infarct volume (approximately 90%) were evident after 48 hours of reperfusion. In ischemic rats that received the same amount of cold saline systemically through a femoral artery, a mild hypothermia was induced only in the cerebral cortex (35.3 ± 0.2°C) and returned to normal within 5 minutes. No significant reductions in infarct volume were observed in this group or in the ischemic group with local warm saline infusion or without infusion. Furthermore, brain-cooling infusion significantly (P < 0.01) improved motor behavior in ischemic rats after 14 days of reperfusion. This improvement continued for up to 28 days after reperfusion. CONCLUSIONLocal prereperfusion infusion effectively induced hypothermia and ameliorated brain injury from stroke. Clinically, this procedure could be used in acute stroke treatment, possibly in combination with intra-arterial thrombolysis or mechanical disruption of clot by means of a microcatheter.

Impaired motor learning and diffuse axonal damage in motor and visual systems of the rat following traumatic brain injury

Abstract Cognitive-motor functioning or motor skill learning is impaired in humans following traumatic brain injury. A more complete understanding of the mechanisms involved in disorders of motor skill learning is essential for any effective rehabilitation. The specific goals of this study were to examine motor learning disorders, and their relationship to pathological changes in adult rats with mild to moderate closed head injury. Motor learning deficits were determined by comparing the ability to complete a series of complex motor learning tasks with simple motor activity. The extent of neuronal damage was determined using silver impregnation. At all post-injury time points (day 1 to day 14), statistically significant deficits were observed in parallel bar traversing, foot placing, ladder climbing, and rope climbing. Performance improved with time, but never reached control levels. In contrast, no deficits were found in simple motor activity skills tested with beam balance and runway traverse. Histologically, axonal degeneration was widely distributed in several brain areas that relate to motor learning, including the white matter of sensorimotor cortex, corpus callosum, striatum, thalamus and cerebellum. Additionally, severely damaged axons were observed in the primary visual pathway, including the optic chiasm, optic tract, lateral geniculate nuclei, and superior colliculus. These findings suggest that motor learning deficits could be detected in mild or moderate brain injury, and this deficit could be attributed to a diffuse axonal injury distributed both in the motor and the visual systems. [Neurol Res 2001; 23: 193-202]

A cell junction pathology of neural stem cells leads to abnormal neurogenesis and hydrocephalus

Most cells of the developing mammalian brain derive from the ventricular (VZ) and the subventricular (SVZ) zones. The VZ is formed by the multipotent radial glia/neural stem cells (NSCs) while the SVZ harbors the rapidly proliferative neural precursor cells (NPCs). Evidence from human and animal models indicates that the common history of hydrocephalus and brain maldevelopment starts early in embryonic life with disruption of the VZ and SVZ. We propose that a cell junction pathology involving adherent and gap junctions is a final common outcome of a wide range of gene mutations resulting in proteins abnormally expressed by the VZ cells undergoing disruption. Disruption of the VZ during fetal development implies the loss of NSCs whereas VZ disruption during the perinatal period implies the loss of ependyma. The process of disruption occurs in specific regions of the ventricular system and at specific stages of brain development. This explains why only certain brain structures have an abnormal development, which in turn results in a specific neurological impairment of the newborn. Disruption of the VZ of the Sylvian aqueduct (SA) leads to aqueductal stenosis and hydrocephalus, while disruption of the VZ of telencephalon impairs neurogenesis. We are currently investigating whether grafting of NSCs/neurospheres from normal rats into the CSF of hydrocephalic mutants helps to diminish/repair the outcomes of VZ disruption.

The microglial response to progressive hydrocephalus in a model of inherited aqueductal stenosis

Although gliosis has been reported to be a common and persistent feature in the white matter of hydrocephalic brains, no studies have identified the cell types that characterize this response. Therefore, the present study has employed histochemical methods to evaluate microglial cells in the brains of infant rats with inherited hydrocephalus. This strain of rats acquires hydrocephalus during late fetal stages due to aqueductal stenosis. Tissue from the sensorimotor and auditory cortices of 12- and 21-day-old hydrocephalic and normal H-Tx rats was processed and stained for the lectin microglial marker Griffonia simplicifolia (GSA-IB4). During the progression of hydrocephalus, GSA-positive cells exhibited three changes: (1) Cytologically, the cell bodies were enlarged, and their processes were thicker, longer and more numerous. These changes were most notable in the gray matter. (2) The packing density of GSA-positive cells was either increased or decreased, depending on the age of the animal and the severity of hydrocephalus. (3) Localized clusters of GSA-positive cells were conspicuous in the white matter of 12-day animals with mild hydrocephalus, and in the gray matter of 21-day animals with severe hydrocephalus. These results indicate that the microglial response is initiated during intermediate stages of hydrocephalus, and is not restricted to the periventricular white matter. These changes may signal other pathophysiologic events in the hydrocephalic brain, and demonstrate that microglia constitute one important element in the gliosis that accompanies hydrocephalus.

Decorin prevents the development of juvenile communicating hydrocephalus

In post-haemorrhagic and other forms of communicating hydrocephalus, cerebrospinal fluid flow and drainage is obstructed by subarachnoid fibrosis in which the potent fibrogenic cytokine transforming growth factor-β has been aetiologically implicated. Here, the hypothesis that the transforming growth factor-β antagonist decorin has therapeutic potential for reducing fibrosis and ventriculomegaly was tested using a rat model of juvenile communicating hydrocephalus. Hydrocephalus was induced by a single basal cistern injection of kaolin in 3-week-old rats, immediately followed by 3 or 14 days of continuous intraventricular infusion of either human recombinant decorin or phosphate-buffered saline (vehicle). Ventricular expansion was measured by magnetic resonance imaging at Day 14. Fibrosis, transforming growth factor-β/Smad2/3 activation and hydrocephalic brain pathology were evaluated at Day 14 and the inflammatory response at Days 3 and 14 by immunohistochemistry and basic histology. Analysis of ventricular size demonstrated the development of hydrocephalus in kaolin-injected rats but also revealed that continuous decorin infusion prevented ventricular enlargement, such that ventricle size remained similar to that in intact control rats. Decorin prevented the increase in transforming growth factor-β1 and phosphorylated Smad2/3 levels throughout the ventricular system after kaolin injection and also inhibited the deposition of the extracellular matrix molecules, laminin and fibronectin in the subarachnoid space. In addition, decorin protected against hydrocephalic brain damage inferred from attenuation of glial and inflammatory reactions. Thus, we conclude that decorin prevented the development of hydrocephalus in juvenile rats by blocking transforming growth factor-β-induced subarachnoid fibrosis and protected against hydrocephalic brain damage. The results suggest that decorin is a potential clinical therapeutic for the treatment of juvenile post-haemorrhagic communicating hydrocephalus.