Jos Van Reempts

Neocortical localization of tactile/proprioceptive limb placing reactions in the rat

The present study was aimed at delineating the neocortical substrate of tactile/proprioceptive limb placing reactions in rats by means of behavioral tests that excluded the participation of facial stimuli in limb function. Using a photochemical technique, we made unilateral focal lesions in the frontal and parietal neocortex. Fore- and/or hindlimb placing deficits resulted from damage to a fronto-parietal region lying between the medial agranular cortex and the primary somatosensory (whisker barrel field) cortex. When the antero-posterior coordinate was varied from 4 mm anterior to 1 mm posterior to bregma, tactile/proprioceptive forelimb dysfunction was more pronounced after damage to the parietal forelimb area, but lesions confined to the frontal lateral agranular cortex also yielded clear-cut forelimb placing deficits. Damage to either area alone allowed for partial recovery of forelimb function. However, following combined, total destruction of both frontal and parietal forelimb areas, forelimb deficits did not recover. This resembled the irreversible hindlimb deficits after near-total destruction of the parietal hindlimb area. Damage to the medial agranular cortex left limb placing intact. Likewise, for as long as the medial edge of lesions to the whisker barrel field did not come closer than 3 mm to the midline, thus remaining outside the parietal hindlimb area, limb placing remained normal. This sharp medial and lateral delineation of the cortical substrate subserving tactile/proprioceptive limb placing coincides with the borders of a thick, dense subfield of large pyramidal neurons in the deeper parts of layer V. Limb placing remained intact when medial agranular cortex lesions damaged only 30% of that subfield, whereas 70% destruction of that layer following more laterally placed lesions in the parietal hindlimb area produced irreversible hindlimb dysfunction. The severity of hindlimb placing deficits was related to the amount of incursion by whisker barrel field lesions into the subfield of deep layer V large pyramidal neurons. Finally, very large lesions of the occipital cortex did not affect tactile/proprioceptive limb placing. We discuss the neocortical areal and laminar specificity of tactile/proprioceptive limb function in the context of recent neuroanatomical and electrophysiological findings, and their relevance to normal cortical function, recovery from neocortical stroke (including diaschisis), and age-related cortical dysfunction.

Impaired autoregulation of cerebral blood flow in an experimental model of traumatic brain injury.

In order to study the pathophysiology and the intracranial hemodynamics of traumatic brain injury, we have developed a modified closed-head injury model of impact-acceleration that expresses several features of severe head injury in humans, including acute and long-lasting intracranial hypertension, diffuse axonal injury, neuronal necrosis, bleeding, and edema. In view of the clinical relevance of impaired autoregulation of cerebral blood flow after traumatic brain injury, and aiming at further characterization of the model, we investigated the autoregulation efficiency 24 h after experimental closed-head injury. Cortical blood flow was continuously monitored with a laser-Doppler flowmeter, and the mean arterial blood pressure was progressively decreased by controlled hemorrhage. Relative laser-Doppler flow was plotted against the corresponding mean arterial blood pressure, and a two-line segmented model was applied to determine the break point and slopes of the autoregulation curves. The slope of the curve at the right hand of the break point was significantly increased in the closed head injury group (0.751 +/- 0.966%/mm Hg versus -0.104 +/- 0.425%/mm Hg,p = 0.028). The break point tended towards higher values in the closed head injury group (62.2 +/- 20.8 mm Hg versus 46.9 +/- 12.7 mm Hg; mean +/- SD, p = 0.198). It is concluded that cerebral autoregulation in this modified closed head injury model is impaired 24 h after traumatic brain injury. This finding, in addition to other characteristic features of severe head injury established earlier in this model, significantly contributes to its clinical relevance.

The hypoxic brain: Histological and ultrastructural aspects

A brief review of structural damage to cerebral cells resulting from experimentally induced hypoxia or ischemia is presented. The histological aspect of the brain is compared in different animal models with respect to the onset and progression of damage. Cell changes detected in the early post-hypoxic period consist of microvacuolation and seem to be fully reversible. Coagulative cell change and edematous cell change which may be considered as the morphologic equivalent of irreversible cell death, develop in a later phase, often as a result of secondary events such as microcirculatory impairment or tissue lactic acidosis. A striking difference in vulnerability exists between cerebral cell types or anatomic brain regions. Possible determinant factors for this phenomenon are discussed. Finally, the special contribution of calcium in cell destructive processes is demonstrated with the aid of ultrastructural calcium distribution studies.

Dorsal-ventral gradient in vulnerability of CA1 hippocampus to ischemia: a combined histological and electrophysiological study.

Transverse hippocampal slices were prepared after 7 days survival from rats subjected to 8 min of global incomplete ischemia by temporary occlusion of both carotid arteries and hypotension. The slices demonstrated a dorsal-ventral gradient in the amount of ischemic neuronal necrosis in the CA1 region. Histologically ischemic cell change decreased from 90% dorsoseptally to 10% ventrotemporally. Electrophysiological analysis of the number of slices with viable synaptic transmission in CA1 also revealed a septotemporal gradient in susceptibility to ischemia.

A new method to study activated oxygen species induced damage in cardiomyocytes and protection by Ca2+-antagonists

It has been proposed that oxygen derived free radicals contribute to reperfusion injury in ischemic tissue: radical induced lipid peroxidation is believed to cause membrane destruction, eventually evolving to cell death. A method is introduced which investigates the effect of exogenously generated reactive O2 species on isolated Ca2+-tolerant rat cardiomyocytes. Singlet oxygen (O2(1)), generated by photo-excitation of the photosensitive dye rose bengal, induced the transformation of elongated rod-shaped cells into hypercontracted rounded cardiomyocytes. These shape changes were prevented by removal of extracellular Ca2+ or by addition of radical scavengers. Pre-treatment with various classes of Ca2+-antagonists dose-dependently reduced the number of hypercontracted cardiomyocytes after exposure to O2(1). Compounds not active on the slow Ca2+-channel (e.g. flunarizine-like) provided a better degree of protection than the genuine slow Ca2+-channel blockers (e.g. dihydropyridines). Ultrastructurally, cardiomyocytes exposed to O2(1) showed a loss of cytochemically demonstrable sarcolemma-associated Ca2+ and the presence of clustered Ca2+-deposits in the mitochondria. Drug pre-treated cells displayed a Ca2+-distribution pattern comparable to unchallenged control cells.

Ischemic brain injury and cell calcium: morphologic and therapeutic aspects.

Histopathological data obtained from different experimental models of hypoxia and ischemia were evaluated in order to extend current knowledge of mechanisms responsible for delayed neuronal cell death. Special attention is given to the distribution of calcium (Ca2+) in vulnerable areas during the postischemic period. Between an initial defensive Ca2+ sequestration, which is completely reversible, and final toxic Ca2+ overload, which is associated with irreversible neuronal necrosis, important Ca2+ shifts could be demonstrated cytochemically. Such shifts occur mainly at excitatory presynaptic sites and seem to precede structural ischemic cell change in postsynaptic areas. Recent results obtained with some Ca2+ entry blockers indicate that prophylactic treatment and postischemic intervention prevent cytosolic Ca2+ overload and reduce delayed brain injury.

Effects of asphyxia on the fetal lamb brain

OBJECTIVE Our purpose was to study the effect of fetal asphyxia on the release of hypoxanthine and xanthine in cerebrospinal fluid and on brain histologic characteristics. STUDY DESIGN In seven fetal lambs (3 to 5 days after surgery, gestational age 124.3 +/- 2.6 days) asphyxia was induced by restriction of uterine blood flow. RESULTS Fetal pH and base excess were reduced to 6.99 +/- 0.02 and -17.6 +/- 0.9 mmol/L, respectively. Cerebral blood flow increased during asphyxia and returned to normal in the recovery phase. Maximum concentrations of cerebrospinal fluid hypoxanthine and xanthine were reached in the normoxemic recovery phase. This high level of substrates during normoxemia facilitates oxygen free radical formation and may thus aggravate postasphyctic brain damage. Histologic evaluation of the brain 3 days after the insult showed a variable degree of edema. Coagulative neuronal changes, characteristic of irreversible cell death, were only occasionally detected. These changes were most obvious in the Purkinje cells of the cerebellum. CONCLUSIONS Fetal asphyxia induced by uterine blood flow restriction is associated with high levels of cerebrospinal fluid hypoxanthine and xanthine in the recovery phase. Microscopically detectable brain damage, although not extensive, is mainly located in the cerebellum.

Altered Na+-channel function as an in vitro model of the ischemic penumbra: action of lubeluzole and other neuroprotective drugs

Veratridine blocks Na(+)-channel inactivation and causes a persistant Na(+)-influx. Exposure of hippocampal slices to 10 microM veratridine led to a failure of synaptic transmission, repetitive spreading depression (SD)-like depolarizations of increasing duration, loss of Ca(+)-homeostasis, a large reduction of membrane potential, spongious edema and metabolic failure. Normalization of the amplitude of the negative DC shift evoked by high K+ ACSF 80 min after veratridine exposure was taken as the primary endpoint for neuroprotection. Compounds whose mechanisms of action includes Na(+)-channel modulation were neuroprotective (IC50-values in microM): tetrodotoxin 0.017, verapamil 1.18, riluzole 1.95, lamotrigine > or = 10, and diphenylhydantoin 16.1. Both NMDA (MK-801 and PH) and non-NMDA (NBQX) excitatory amino acid antagonists were inactive, as were NOS-synthesis inhibitor (nitro-L-arginine and L-NAME) Ca(2+)-channel blockers (cadmium, nimodipine) and a K(+)-channel blocker (TEA). Lubeluzole significantly delayed in time before the slices became epileptic, postponed the first SD-like depolarization, allowed the slices to better recover their membrane potential after a larger number of SD-like DC depolarizations, preserved Ca2+ and energy homeostasis, and prevented the neurotoxic effects of veratridine (IC50-value 0.54 microM). A concentration of lubeluzole, which was 40 x higher than its IC50-value for neuroprotection against veratridine, had no effect on repetitive Na(+)-dependent action potentials induced by depolarizing current in normal ACSF. The ability of lubeluzole to prevent the pathological consequences of excessive Na(+)-influx, without altering normal Na(+)- channel function may be of benefit in stroke.

Synaptic plasticity in rat hippocampus associated with learning

Rats subjected to a one-way active avoidance task consisting of 3 daily training sessions, showed obvious shape changes in dendritic spines of the hippocampal supragranular molecular layer. Performance, expressed as the number of avoidances per 10 trials, significantly improved in the second and third session (P < 0.001). In trained animals, at the end of the third session, the amount of perforated concave synapses significantly increased as compared to untrained controls (P < 0.05). When compared with a group of sham-shocked rats, the increase was less pronounced. The length of the postsynaptic density in both, perforated and non-perforated synapses, significantly increased in comparison with untrained control and sham-shocked animals (perforated: P < 0.005; non-perforated: P < 0.05). The results are indicative for the existence of synaptic remodeling and turnover in rats subjected to one-way active avoidance training.

Protection With Lubeluzole Against Delayed Ischemic Brain Damage in Rats: A Quantitative Histopathologic Study

BACKGROUND AND PURPOSE Cerebral ischemia may lead to glutamate-induced excitotoxic damage in vulnerable brain areas. Lubeluzole is not an N-methyl-D-aspartate antagonist but prevents postischemic increase in extracellular glutamate concentrations. The present study examined whether lubeluzole, administered after global incomplete ischemia in rats, is capable of preserving the structural integrity of CA1 hippocampus. METHODS Ischemia was induced by bilateral carotid artery occlusion and severe hypotension for a duration of 9 minutes. Delayed neuronal cell death was histologically evaluated 7 days later. This was done by scoring acidophilic cell change and coagulative necrosis and by counting the number of surviving neurons in the CA1 subfield. Experiments were performed according to a paired design (13 animals per treatment group). RESULTS Posttreatment with lubeluzole (0.31 mg/kg i.v. bolus at 5 minutes and 0.31 mg/kg i.v. infusion during 1 hour) resulted in significant neuroprotection. Whereas in the untreated rats there were 42 (median) viable neurons per millimeter CA1 layer in the left and 69 in the right hemisphere, in the drug-treated rats 99 viable neurons per millimeter were found in the left (P = .002) and 113 in the right hemisphere (P = .013). Histological scores, reflecting altered staining properties of the hippocampal cells, correlated strongly with the quantitative data, reflecting the structural integrity of CA1 pyramidal neurons. CONCLUSIONS Lubeluzole, when administered after an ischemic insult in rats, protects vulnerable brain regions against delayed structural injury. The results support the potential clinical use of this new drug in stroke treatment.