Garnette Sutherland

Ischemia-induced cellular redistribution of the astrocytic gap junctional protein connexin43 in rat brain

The distribution and levels of the astrocytic gap junction protein, connexin43 (Cx43) was analyzed in various regions of brain as a function of time after neuronal loss and consequent reactive gliosis induced by bilateral carotid occlusion in rats. In the striatum 2 days after induction of ischemia, immunostaining intensity for Cx43 increased in animals exhibiting mild to moderate striatal damage, whereas areas of reduced staining surrounded by elevated levels of Cx43 immunoreactivity were observed in animals with severe ischemic damage. Immunolabelling of glial cell bodies was evident in ischemic, but not normal, striatum. Similar, though less dramatic, changes were seen at 7 days post-ischemia. Compared with the fine punctate pattern of Cx43 staining seen in normal striatum, ischemic striatal areas contained large aggregates of punctate profiles. In the hippocampus, increased immunostaining was seen at 2 and 7 days post-ischemia and, unlike normal hippocampus, neurons in the CA3 pyramidal cell layer were surrounded by a network of Cx43-immunoreactive puncta at the latter survival time. Immuno-EM analysis of ischemic tissue revealed numerous immunolabelled gap junctions among astrocytic processes in the vicinity of degenerating neurons and elevated levels of intracellular Cx43 immunoreactivity in astrocytic processes and cell bodies. No differences in protein levels or phosphorylation states of Cx43 were detected in either hippocampus or striatum by Western blot analyses of ischemic and control tissue. These results suggest that astrocytes respond to an ischemic insult by reorganizing their gap junctions, that the qualitative nature of their response is dependent on the severity of neuronal damage or loss, and that a pool of Cx43 normally undetectable by immunohistochemistry may contribute to the ischemia-induced elevations of immunolabelling for this protein.

Three openings of the blood-brain barrier produced by forebrain ischemia in the rat.

A sensitive radiotracer method was used to explore the time course and regional pattern of blood-brain barrier (BBB) opening that occurs in a rat forebrain ischemia model that mimics temporary cardiac arrest. Immediately following 10 min of ischemia, transfer constants (Ki) for blood to brain permeation of [3H]sucrose were augmented severalfold, indicating widespread BBB opening. After 6 h, a delayed intensification of opening was evident in striatum and hippocampus, regions known to undergo selective, delayed neuronal death. There was generalized BBB recovery by 24 h except in experiments that involved prolonged ischemia (25 min) or concomitant brain hyperthermia (41 degrees C, 10 min). These protocols evoked a third opening; a marked upward increment in Ki and % H2O developed in neocortex between 6 and 24 h post-ischemia. Pharmacological or other manipulations of these temporal and regional patterns of altered transfer constants may aid understanding of the interplay between microvascular damage, edema, and neuronal death following brain ischemia.

The effects of caffeine on ischemic neuronal injury as determined by magnetic resonance imaging and histopathology.

The effects of caffeine on ischemic neuronal injury were determined in rats subjected to forebrain ischemia induced by bilateral carotid occlusion and controlled hypotension (50 mmHg for 10 min). High resolution (100 microns) multi-slice, multi-echo magnetic resonance images were obtained daily for three consecutive days post-operatively in sham-operated rats and in rats that received either saline vehicle (controls), a single i.v. injection of 10 mg/kg caffeine 30 min prior to an ischemic insult (acute caffeine group), or up to 90 mg/kg per day of caffeine for three consecutive weeks prior to an ischemic insult (chronic caffeine group). Rats in the control group exhibited enhanced magnetic resonance image intensity in the striatum 24 h after ischemia which increased in the striatum and also appeared in the hippocampus after 48 h, and which began to resolve in both regions by 72 h post-ischemia. Histopathological analysis of each rat following the final magnetic resonance examination showed that ischemic neuronal injury was strictly confined to the brain regions showing magnetic resonance image changes. Acute caffeine rats showed accelerated changes in the magnetic resonance images, with increased hippocampal intensity appearing at 24 h post-ischemia. Although there was magnetic resonance evidence of accelerated injury, quantitative analysis of the histopathological data at 72 h showed no significant difference in the extent of neuronal injury in any brain region between control-ischemic and acute caffeine rats. Nine out of 11 rats in the chronic caffeine group showed no magnetic resonance image changes over the three study days. Chronic caffeine rats had significantly less neuronal damage in all vulnerable brain regions than either of the other groups of ischemic rats. The accelerated ischemic injury in rats treated with an acute dose of caffeine may occur secondary to antagonism of adenosine receptors, whereas protection from ischemic injury following chronic administration of caffeine may be mediated by up-regulation of adenosine receptors.

Anatomic and cardiopulmonary responses to trauma with associated blunt chest injury.

Myocardial dysfunction may result from severe trauma. Therefore, left (LV) and right ventricular (RV) function were prospectively assessed by ECG-gated blood pool radionuclide (RN) angiography in 25 consecutive patients who had sustained severe trauma including blunt chest injuries. Focal abnormalities of RV and LV wall motion were defined in 17 patients; 12, RV; two, LV; and three, biventricular. In two patients traumatic tricuspid insufficiency was also demonstrated, and subsequently verified by contrast angiography. Other means to detect myocardial contusion (enzymatic, electrocardiographic, and Tc-99m pyrophosphate scintigraphy) proved to be insensitive when compared to RN angiography. Two of the five deaths in the group were attributed to refractory arrhythmias. Surgical or post-mortem evidence of traumatic myocardial injury was obtained in five instances when RN angiography indicated contusion. Of the 13 patients available for followup examinations, 11 showed complete or partial resolution of the abnormality and two were unchanged. Comprehensive cardiopulmonary monitoring revealed an inverse relationship between right ventricular ejection fraction (RVEF) and pulmonary vascular resistance (PVR) (R2 = 0.42; p < 0.01) and between the PVR and left ventricular ejection fraction (LVEF) (R2 = 0.48; p < 0.01) and left ventricular end-diastolic volume (LVEDV) (R2 = 0.69; p < 0.01). Further, as right ventricular end-diastolic volume (RVEDV) was increased in trauma, left ventricular function and compliance were reduced. In blunt chest trauma RV contusion occurs more frequently than previously recognized and positive RN angiography constitutes prima facie evidence of direct myocardial injury. Further, LV function remains preload dependent, but may be depressed by elevated PVR impeding the blood flow from RV to LV and/or decreases in LV compliance.

Effect of U74006F on forebrain ischemia in rats.

We examined the effect of a putative lipid peroxidation inhibitor, the 21-aminosteroid U74006F, on transient forebrain ischemia in rats. Acute-treatment rats received either 3 mg/kg U74006F (n = 7) or carrier vehicle (n = 5) intravenously 30 minutes before ischemia, sustained-treatment rats received the same treatment before ischemia followed by 3 mg/kg U74006F (n = 6) or carrier vehicle (n = 5) intraperitoneally every 6 hours for 48 hours, and control rats (n = 7) received no injection. Coronal magnetic resonance images were obtained daily for 3 days, followed by the histological examination of perfusion-fixed brains. Control rats demonstrated magnetic resonance image changes indicative of neuronal damage in the striatum at 24 hours postischemia, followed by changes in the hippocampus and neocortex at 48 hours. No significant effect of U74006F treatment on striatal or hippocampal injury was demonstrated. However, both the acute and sustained U74006F treatments produced a significant reduction in the severity of neuronal damage in the neocortex (p less than 0.05). Our results suggest that U74006F is of benefit in ameliorating ischemic neuronal injury, particularly in the neocortex, and raise the possibility of regional variability in lipid peroxidation following an ischemic insult.

Effect of mannitol, nimodipine, and indomethacin singly or in combination on cerebral ischemia in rats.

The effects of mannitol, nimodipine, and indomethacin on ischemic neuronal injury were examined in 45 rats divided equally into nine groups subjected to 10 minutes of forebrain ischemia. Of two control groups, one received maintenance fluids while the other received a normal saline bolus. In the remaining seven groups, mannitol, nimodipine, and indomethacin were administered singly or in combination 5 minutes before forebrain ischemia. Seven days after ischemia, the brains were perfusion-fixed, sectioned coronally into 2.8-mm slices, and stained with hematoxylin and eosin. Ischemic neurons were directly counted on predetermined regions of standardized serial sections. Considerable amelioration of ischemic injury (ischemic neurons/total neurons) was observed with mannitol (ischemic injury, 7 +/- 5% in the hippocampal CA1/CA2 sectors and 28 +/- 17% in the CA3 sector). This is in contrast to control values of 64 +/- 11% and 80 +/- 6%, respectively, and those obtained in the normal saline group of 70 +/- 10% and 59 +/- 13%, respectively. The beneficial effect with nimodipine reached significance in only the hippocampal CA3 sector (ischemic injury, 35 +/- 21%). Indomethacin showed no significant benefit. Combining the agents resulted in significantly reduced neuronal injury compared with control groups, although the effect was not greater than that achieved with mannitol alone. The degree of ischemic injury was least when all three agents were used in combination (ischemic injury, 12 +/- 12% in the hippocampal CA1/CA2 sectors and 4 +/- 4% in the CA3 sector). Our data support the concept that successfully blocking the ischemic cascade with a single, diversely acting agent or multiple agents will evoke the best beneficial response.

Biological and methodological implications of prostaglandin involvement in mouse brain lipid peroxidation measurements

Enhanced cyclooxygenase-mediated prostaglandin (PG) turnover occurring during sacrifice and biochemical processing of tissues also generates malondialdehyde (MDA), a product of lipid peroxidation (LPO). Studies reporting on LPO estimated by thiobarbituric acid reactive substances (TBARS) have failed to consider such artefactual increases. This study reports the relative proportion of PG metabolism-derived MDA (PG-MDA) in mouse brain regions during the TBARS assay. The cyclooxygenase inhibitor indomethacin significantly lowered MDA in fronto-parietal cortex and corpus striatum. Indomethacin (50–800 μg/ml, in vitro) increased estimated TBARS in whole brain. Such enhancement was absent when indomethacin (20–80 μg/sample) was added to the MDA standard curve, reflecting its interaction with TBARS other than MDA. PG-MDA contributes as much as 15% to the total estimated value of MDA in fronto-parietal cortex and corpus striatum and must be corrected for in LPO studies.

Effect of sustained pyridoxine treatment on seizure susceptibility and regional brain amino acid levels in genetically epilepsy-prone BALB/c mice

Summary: Epilepsy‐prone and epilepsy‐resistant substrains were selectively bred from a strain of BALB/c mice; audiogenic‐sensitive epilepsy‐prone animals showed enhanced sensitivity to chemical convulsants. Treatment with pyridoxine (100 mg/L in drinking water) initiated at mating and continued throughout pregnancy and the life of the offspring abolished the enhanced sensitivity to chemical convulsants and reduced the severity of audiogenic seizures. Withdrawal of pyridoxine restored the enhanced seizure sensitivity. [1H] Nuclear magnetic resonance (NMR) spectroscopy of perchloric acid extracts of tissue was used to determine the concentrations of several compounds [N‐acetylaspartate (NAA), GABA, glutamate, aspartate, alanine, taurine, creatine, cholines, inositol] in the hippocampus, neocortex, brainstem, and cerebellum of untreated and pyridoxine‐treated 6‐week‐old female animals. The ratios of the concentrations of excitatory to inhibitory putative neurotransmitteramino acids tended to be higher in epilepsy‐prone animals, with the most pronounced difference being a significantly elevated glutamate/GABA ratio in every brain region examined. Pyridoxine treatment abolished this imbalance in the hippocampus, brainstem, and cerebellum, but not in the neocortex. Treatment of epilepsy‐resistant animals with pyridoxine using the same protocol decreased the glutamate/GABA concentration ratio in the hippocampus, brainstem, and neocortex and resulted in impaired development of the animals. The amino acid imbalance and the accompanying seizure susceptibility in these genetically epilepsy prone mice may originate from an inborn error in pyridoxine metabolism or in a pyridoxine‐dependent enzyme system.

Autonomic Neuropathy in BB Rats and Alterations in Bladder Function

In vivo urinary bladder function was examined in BB rats after 4 and 6 mo of diabetes, and the data were correlated with morphometric changes in the pelvic and hypogastric nerves, which constitute the micturition reflex arc. After controlled bladder distension, diabetic animals revealed irregular bladder contractions at frequencies that were reduced to 33% of normal values and with significantly increased amplitudes. The abnormal micturition in diabetic animals was elicited at moderately elevated threshold volumes. These functional abnormalities of the diabetic bladder were associated with a progressive axonopathy of afferent myelinated sensory fibers and later-occurring axonal atrophy of unmyelinated efferent preganglionic fibers. These data suggest that diabetic urinary bladder dysfunction is initiated by a visceral sensory neuropathy involving the afferent limb of the micturition reflex arc.

Effect of lidocaine on forebrain ischemia in rats.

We examined the effect of lidocaine on ischemic neuronal injury in the rat forebrain ischemia model. Cerebral ischemia was achieved with bilateral carotid artery occlusion and controlled hypotension to a mean of 50 torr for 10 minutes. Perfusion-fixation was performed 7 days after ischemia, subsequent to which the brains were sectioned coronally and stained with hematoxylin and eosin. Ischemic neuronal injury was quantitatively expressed (after direct counting) as a percentage of total neurons, that is, ischemic neurons divided by (ischemic neurons + normal neurons). Predictably, the selectively vulnerable hippocampal areas exhibited the most marked neuronal injury. In the CA1/CA2 sectors, lidocaine-treated rats demonstrated less injury (34 +/- 14%) than untreated (64 +/- 9%) or saline-treated (70 +/- 10%) rats. However, these superficially pronounced numerical differences were not of statistical significance (p greater than 0.05). In the CA3 sector, neuronal injury in lidocaine-treated rats (31 +/- 14%) was significantly different at p less than 0.05 from the untreated (80 +/- 5%) but not the saline-treated (59 +/- 13%) group. We conclude that lidocaine may have an only marginal beneficial effect on forebrain ischemia in rats.