Manuel F. Gonzalez

Heat shock proteins as markers of neural injury.

Systemic or intracerebral injections of kainic acid induced immunoreactivity for the 72 kDa heat shock protein (HSP72) in individual neurons of the rat brain in patterns matching the known histopathology of the particular injury. HSP72 immunostaining was also induced in and around areas of infarction following experimental strokes. These results suggest that HSP72 immunocytochemistry may be used as a marker of cellular injury in the mammalian brain.

Induction of heat shock protein 72-like immunoreactivity in the hippocampal formation following transient global ischemia

Global ischemia was produced in adult rats by combining bilateral carotid artery occlusions with systemic hypotension for 5 or 10 minutes. Induction of the 72 kD heat shock protein (HSP72) in the hippocampus was examined immunocytochemically 18-24 hours later. Several patterns of HSP72-like immunoreactivity (HSP72LI) were observed. Five minutes of ischemia induced HSP72 in isolated columns of CA1a pyramidal neurons, or throughout CA1 pyramidal neurons and dentate hilar neurons. Ten minutes of ischemia induced marked HSP72LI in CA3 pyramidal neurons, moderate HSP72LI in dentate granule cells, and minimal HSP72LI in CA1 pyramidal, dentate hilar neurons, and hippocampal glia. Two hippocampi subjected to 10 minutes of ischemia exhibited marked HSP72LI in capillary endothelial cells but no neuronal or glial HSP72LI. It is proposed that (a) the induction of HSP72 in hippocampal sectors correlates with their vulnerability to global ischemia (CA1 greater than hilus greater than CA3 greater than dentate gyrus); (b) the induction of HSP72 in hippocampal cells correlates with their vulnerability to global ischemia in that mild ischemia induced HSP72 only in neurons, moderate ischemia in neurons and glia, and severe ischemia only in capillary endothelial cells; (c) the failure to induce HSP72 in hippocampal neurons in 2 cases of 10 min ischemia may be related to severe injury causing disruption of protein synthesis in these cells.

Induction of the c-fos gene product in rat forebrain following cortical lesions and NGF injections.

Neocortical lesions and NGF injections into neocortex induce the immunostaining of Fos, the c-fos gene product, in neuronal nuclei in ipsilateral cortex, and amygdala. Adjacent structures including hippocampus, septal nuclei, globus pallidus, and thalamus were unaffected. It is hypothesized that trophic molecules or other chemicals are released at the injury site and these induce the c-fos gene in cells throughout the ipsilateral hemisphere. Fos induction might mediate metabolic or plasticity responses to the focal injury.

Trigeminal nerve section induces Fos-like immunoreactivity (FLI) in brainstem and decreases FLI in sensory cortex.

Transecting the infraorbital nerve to the rat whiskers induced Fos-like immunoreactivity (FLI) in lamina I and II neuronal nuclei of the spinal trigeminal nucleus pars caudalis (Sp5c). The Fos-like immunostaining persisted for several weeks. The prolonged expression of FLI in Sp5c could be related to persistent activity in the sectioned nerve, or to trophic effects of injured ganglion neurons on brainstem cells. We postulate that Fos and related proteins may be involved in mediating alterations in gene expression associated with relatively long-term CNS adaptations to peripheral nerve injuries. Surprisingly, FLI decreased in contralateral sensory cortex, mainly in layers 2, 3 and 6, up to several days after the lesion. These decreases of cortical FLI may be due to decreased sensory neuronal activity, and/or to reducing the trophic influence of thalamic inputs on cortical neurons.

Fetal frontal cortex transplanted to injured motor/sensory cortex of adult rats: reciprocal connections with host thalamus demonstrated with WGA-HRP

Fetal frontal cortex was transplanted into lesion cavities formed in host motor/sensory cortex of adult rats. Eight to twenty-eight weeks later wheat germ agglutinin conjugated with horseradish peroxidase (WGA-HRP) was injected into host thalamus and the brain was sectioned and reacted using a sensitive TMB procedure. A large amount of fine granular WGA-HRP was detected in most transplants. This could represent anterograde transport demonstrating that injured adult host thalamic neurons sprouted axons into fetal cortical transplants. Conversely, none or very few retrogradely labeled pyramidal neurons were present in the transplants. This indicates that pyramidal neurons in transplants either did not sprout into adult host brain or sprouted such short distances that they did not pick up the WGA-HRP. These results are compatible with the hypothesis that high trophic/growth factor levels in newborn or fetal brain and low levels in adults determine the more extensive connections seen in newborn hosts compared with those in adult transplanted hosts. The data are also consistent with the proposal that adult host brains impair axonal growth. Functionally, the data suggest that although corticofugal effects of fetal cortical transplants in adult host brains are likely to be limited, transplants could exert beneficial trophic effects on adult host thalamic neurons.

Fetal cortical transplants ameliorate thalamic atrophy ipsilateral to neonatal frontal cortex lesions

Thalamic atrophy develops ipsilateral to neonatal frontal cortex lesions. Fetal cortex transplants placed in these lesions at birth ameliorate the thalamic atrophy. This could be due to trophic effects of the transplants on the neonatal host thalamus, host cortex, or both.

Fetal frontal cortex transplant (14C) 2‐deoxyglucose uptake and histology: Survival in cavities of host rat brain motor cortex

Fetal frontal neocortex from 18-day-old rat embryonic brain was transplanted into cavities in 30-dayold host motor cortex. Sixty days after transplantation, 5 of 15 transplanted rats had surviving fetal transplants. The fetal cortex transplants were physically attached to the host brain, completely filled the original cavity, and had numerous surviving cells including pyramidal neurons. Cell lamination within the fetal transplant was abnormal. The (14C) 2-deoxyglucose uptake of all five of the fetal neocortex transplants was less than adjacent cortex and contralateral host motor-sensory cortex, but more than adjacent corpus callosum white matter. The results indicate that fetal frontal neocortex can be transplanted into damaged rat motor cortex. The metabolic rate of the transplants suggests they could be partially functional.

Injured adult neocortical neurons sprout fibers into surviving fetal frontal cortex transplants: Evidence using NADPH-diaphorase staining

Abstract NADPH-diaphorase-stained neurons located in injured adult rat brain motor cortex sprout fibers into surviving fetal frontal cortex transplants.

Whisker stimulation metabolically activates thalamus following cortical transplantation but not following cortical ablation

Local cerebral glucose utilization was assessed during whisker stimulation by 2-deoxyglucose autoradiography. Whisker stimulation increased local cerebral glucose utilization in brainstem, thalamus and whisker sensory cortex in normal rats. Whereas whisker stimulation increased glucose metabolism in brainstem, whisker stimulation failed to increase glucose metabolism in thalamus of rats that had whisker sensory cortex ablated 5 h to five weeks previously. The failure of whisker stimulation to activate thalamus after cortical ablations was probably not due to decreased cortical input to thalamus because whisker stimulation activated thalamus after large cortical tetrodotoxin injections. Failure of whisker stimulation to activate thalamus at early times (5 h and one day) after cortical ablations was not due to thalamic neuronal death, since it takes days to weeks for axotomized thalamic neurons to die. The failure of whisker stimulation to activate thalamus at early times after cortical ablations was likely due to the failure of trigeminal brainstem neurons that project to thalamus to activate axotomized thalamic neurons. This might occur because of synaptic retraction, glial stripping or inhibition of trigeminal brainstem synapses onto thalamic neurons. The thalamic neuronal death that occurs over the days and weeks following cortical ablations was associated with thalamic hypometabolism. This is consistent with the idea that the thalamic neurons die because of the absence of a cortically derived trophic factor, since the excitotoxic thalamic cell death that occurs following cortical kainate injections is associated with thalamic hypermetabolism. The glucose metabolism of parts of the host thalamus was higher and the glucose metabolism in surrounding nuclei lower than the normal side of thalamus in rats that sat quietly and had fetal cortex transplants placed into cavities in whisker sensory cortex five to 16 weeks previously. Whisker stimulation in these subjects activated the contralateral host thalamus and fetal cortical transplants. This was accomplished using a double-label 2-deoxyglucose method to assess brain glucose metabolism in the same rat while it was resting and during whisker stimulation. The high glucose metabolism of parts of host thalamus ipsilateral to the fetal cortical transplants is consistent with prolonged survival of some axotomized thalamic neurons. The finding that whisker stimulation activates portions of host thalamus further suggests that the cortical transplants maintained survival of the host thalamic neurons and that synaptic connections between whisker brainstem and thalamic neurons were functional.

Motor deficits are produced by removing some cortical transplants grafted into injured sensorimotor cortex of neonatal rats

Fetal frontal cortex was transplanted into cavities formed in the right, motor cortex of neonatal rats. As adults, the animals were trained to press two levers in rapid succession with their left forelimb to receive food rewards. Once they had reached an optimal level of performance, the effect of removing their transplants was assessed. Surgical removal of transplants significantly impaired the performance of 2 of 4 subjects. Placing a crossstrain skin graft to induce the immunological rejection of the transplants produced a behavioral deficit in 1 of 2 subjects with complete transplant removal. Skin grafts produced no behavioral effects in four subjects that had surviving transplants. Since the motor deficit produced by transplant removal resembled those observed following the removal of normal motor cortex, we propose that these three transplants functioned within the host brain. Histology Showed that the procedures used to remove cortical grafts did not injure any host brains. Therefore, host brain damage is unlikely to account for the behavioral deterioration that followed transplant removals.