R. W. Currie

Transgenic mice expressing the human heat shock protein 70 have improved post-ischemic myocardial recovery.

Heat shock treatment induces expression of several heat shock proteins and subsequent post-ischemic myocardial protection. Correlations exist between the degree of stress used to induce the heat shock proteins, the amount of the inducible heat shock protein 70 (HSP70) and the level of myocardial protection. The inducible HSP70 has also been shown to be protective in transfected myogenic cells. Here we examined the role of human inducible HSP70 in transgenic mouse hearts. Overexpression of the human HSP70 does not appear to affect normal protein synthesis or the stress response in transgenic mice compared with nontransgenic mice. After 30 min of ischemia, upon reperfusion, transgenic hearts versus nontransgenic hearts showed significantly improved recovery of contractile force (0.35 +/- 0.08 versus 0.16 +/- 0.05 g, respectively, P < 0.05), rate of contraction, and rate of relaxation. Creatine kinase, an indicator of cellular injury, was released at a high level (67.7 +/- 23.0 U/ml) upon reperfusion from nontransgenic hearts, but not transgenic hearts (1.6 +/- 0.8 U/ml). We conclude that high level constitutive expression of the human inducible HSP70 plays a direct role in the protection of the myocardium from ischemia and reperfusion injury.

Transgenic mice expressing the human inducible Hsp70 have hippocampal neurons resistant to ischemic injury.

Using transgenic mice constitutively expressing the human inducible Hsp70, we examined the role of Hsp70 on cell survival after focal cerebral ischemia. Twenty-four hours after permanent occlusion of the middle cerebral artery, no difference in infarct area was detected between Hsp70-transgenic and non-transgenic mice. In the non-transgenic mice, many pyramidal neurons of the ipsilateral hippocampus were observed to be pyknotic. However, in all Hsp70-transgenic mice, hippocampal pyramidal neurons showed normal morphology and no evidence of pyknosis. This suggests that constitutive expression of Hsp70 reduces the extent of damage following permanent middle cerebral artery occlusion.

Constitutive expression of the 27-kDa heat shock protein (Hsp27) in sensory and motor neurons of the rat nervous system.

In this study, the constitutive expression of the 27‐kDa heat shock protein (Hsp27) in the adult rat central nervous system has been examined by immunohistochemistry and by two‐dimensional gel Western blot analysis. Hsp27 immunoreactivity was observed primarily in motoneurons of cranial nerve nuclei and spinal cord, and in primary sensory neurons and their central processes. Also, Hsp27 immunoreactivity was present in neurons of the arcuate nucleus and of the reticular formation. However, only a subset of these neurons was Hsp27‐ immunoreactive. Most general somatic efferent motoneurons of the hypoglossal nucleus and spinal motor columns and most special visceral efferent motoneurons of the cranial nerve nuclei were Hsp27‐positive. In contrast, fewer general somatic efferent motoneurons for eye muscles were Hsp27‐positive, and only a small proportion of general visceral efferent neurons, i.e., parasympathetic and sympathetic preganglionic neurons, were stained for Hsp27. Many pseudounipolar sensory neurons were Hsp27‐immunoreactive, and the patterns of staining in central sensory nuclei suggested that specific subpopulations of sensory neurons contained Hsp27. The cellular distribution of Hsp27 was uniform throughout the cytoplasm, including the perikaryon, axon and dendrites, the latter often exhibiting varicosities or beading in distal processes. Western blot analyses revealed that at least three phosphorylated isoforms of Hsp27 were present in the spinal cord. These results suggest that constitutively expressed Hsp27 may be related to functional subpopulations of motoneurons and primary sensory neurons. J. Comp. Neurol. 384:409–428, 1997.

Expression of the 27,000 mol. wt heat shock protein following kainic acid-induced status epilepticus in the rat

Western analysis and immunohistochemistry were used to determine the time-course and the distribution of the 27,000 mol. wt heat shock protein, Hsp27, in rat brain following systemic administration of kainic acid. No Hsp27 immunoreactivity was detected in naive control animals or in rats that failed to develop status epilepticus. Hsp27 immunoreactivity was detected as early as 12 h in the parietal cortex, piriform cortex and the hippocampus of rats that developed status epilepticus. The number of cells expressing Hsp27 and the intensity of Hsp27 immunoreactivity were increased 24 h after kainic acid administration. Hsp27 immunoreactivity was still observed seven days post-kainic acid injection. The morphology of the Hsp27-positive cells and double immunofluorescence against Hsp27 and glial fibrillary acidic protein revealed that Hsp27-positive cells were astrocytes. In addition, the distribution of Hsp27 suggested that astrocytic Hsp27 was dependent on excitation-induced metabolic stress rather than the direct effect of kainic acid on astrocytes.

Cortical Application of Potassium Chloride Induces the Low-Molecular Weight Heat Shock Protein (Hsp27) in Astrocytes

Spreading depression induces tolerance to ischemic injury, and ischemic tolerance has been associated with expression of heat shock proteins (Hsp). Here we examine Hsp27 expression after KCl-induced spreading depression. Twenty-minute cortical KCl application induced Hsp27 immunoreactivity in glial fibrillary acidic protein-positive astrocytes of the ipsilateral neocortex. Systemic administration of MK-801 (3 mg/kg) suppressed KCl-induced Hsp27 expression in the parietal cortex. Astrocytes in the posterior cingulate and retrosplenial cortex did not express Hsp27 after KCl application but did express Hsp27 after systemic administration of high dose MK-801 (9 mg/kg). Whereas Hsp27 was usually observed in all layers of the parietal cortex after 5-minute application of KCl, in 2 of 6 rats, Hsp27 was seen in clusters of astrocytes or in astrocytes in the superficial layers I to III of the parietal cortex. We conclude that (1) cortical application of KCl triggered Hsp27 astrocytic expression; (2) astrocytes in the cingulate and retrosplenial cortex responded differently compared with astrocytes of the parietal cortex; (3) Hsp27 expression progressed from small clusters of astrocytes throughout superficial layers of the cortex that joined and recruited astrocytes in deeper layers; (4) several mechanisms induced Hsp27 astrocytic expression. We propose that Hsp27 is involved in spreading depression-induced ischemic tolerance through protection of astrocyte function.

The inducible 70,000 molecular/weight heat shock protein is expressed in the degenerating dentate hilus and piriform cortex after systemic administration of kainic acid in the rat.

Using both immunohistochemistry and in situ hybridization, we examined the rat brain for the expression of the inducible 70,000 mol. wt heat shock protein, Hsp70, at 3,6,12 and 24 h after systemic administration of kainic acid. In contrast to previous reports, the present study demonstrates that neurons in the regions most susceptible to seizure-induced cell death accumulate both Hsp70 messenger RNA and protein. Neurons in the denate hilus and piriform cortex contained Hsp70 messenger RNA at 6 h and protein at 12 h. These neutrons contained little or no Hsp70 messenger RNA or protein at 24 h when the majority of cells in these area were pyknotic. Injured neurons in areas such as the parietal cortex, which are less susceptible to seizure-induced cell death, expressed and maintained high levels of Hsp70 messenger RNA and protein at 12 and 24 h. This work suggest that Hsp70 messenger RNA and protein are rapidly and transiently expressed in dying neurons, and contradicts the notion that Hsp70 only accumulates in injured neurons that survive.

Injury to retinal ganglion cells induces expression of the small heat shock protein Hsp27 in the rat visual system.

Optic nerve transection results in apoptotic cell death of most adult rat retinal ganglion cells that begins at 4 days and leaves few surviving neurons at 14 days post-injury [Berkelaar et al. (1994) J. Neurosci. 14, 4368-4374]. The small heat shock protein Hsp27 has recently been shown to play a role in sensory neuron survival following peripheral nerve axotomy [Lewis et al. (1999) J. Neurosci. 19, 8945-8953]. To investigate the role of Hsp27 in injured CNS sensory neurons, we have studied the induction and cell-specific expression of Hsp27 in rat retinal ganglion cells 1-28 days after optic nerve transection. Immunohistochemical results indicate that Hsp27 is not present at detectable levels in the ganglion cell layer of control (uninjured) or sham-operated control rats. In contrast, Hsp27 is detected in retinal ganglion cells from 4 to 28 days following axotomy. Furthermore, the percentage of surviving retinal ganglion cells that are Hsp27-positive increased over the same time period. Hsp27 is also detected in glial fibrillary acidic protein-positive astrocytes in the optic layer of the superior colliculus from 4 to 28 days after optic nerve transection. These experiments demonstrate that transection of the optic nerve results in the expression of Hsp27 in three distinct regions of the rat visual system: sensory retinal ganglion cells in the eye, glial cells of the optic tract, and astrocytes in the optic layer of the superior colliculus. Hsp27 may be associated with enhanced survival of a subset of retinal ganglion cells, providing evidence of a protective role for Hsp27 in CNS neuronal injury.

Cell specific expression of Hsp70 in neurons and glia of the rat hippocampus after hyperthermia and kainic acid-induced seizure activity

In this study we investigated the time course, cell-type and stress-specific expression of hsp70 mRNA and Hsp70 protein in glial cells and neurons in the rat brain following heat shock treatment and kainic acid-induced status epilepticus. Transcripts for hsp70 were detected in hippocampal homogenates from 1.5 to 6 h following hyperthermia and from 3 to 24 h following kainic acid-induced seizures. In situ hybridization revealed hsp70 mRNA to be region specific and time-dependent following hyperthermia and kainic acid-induced seizures. Western analysis indicated that Hsp70 reached maximal levels at 3 h after hyperthermia and 12 h after kainic acid-induced seizures. Immunohistochemistry revealed low level expression of Hsp70 protein in dentate granule cells at 1.5 and 3 h after hyperthermia. No Hsp70 protein was detected in neurons of the pyramidal cell layer or dentate hilus at any time following hyperthermia. Small Hsp70-immunoreactive cells were detected throughout the hippocampus following hyperthermia that, based on cell size, distribution, and double-labeling with vimentin, were considered to be glia. In contrast, high levels of Hsp70 protein were detected in neurons of the pyramidal cell layer and dentate hilus at 24 h after seizure-inducing kainic acid injection. These results suggest that expression of Hsp70 protein is cell-specific depending on the stressor. In addition, finding high levels of Hsp70 mRNA in the dentate granule cells after hyperthermia, but little or no Hsp70 protein, suggests that the synthesis of the protein is also regulated at the post-transcriptional level following hyperthermia.

Complexity of sensory environment drives the expression of candidate-plasticity gene, nerve growth factor induced-A

Exposure of animals to an enriched environment triggers widespread modifications in brain circuitry and function. While this paradigm leads to marked plasticity in animals chronically or acutely exposed to the enriched environment, the molecular mechanisms that enable or regulate such modifications require further characterization. To this end, we have investigated the expression profiles of both mRNA and protein products of a candidate-plasticity gene, nerve growth factor induced-A (NGFI-A), in the brains of rats exposed to increased environmental complexity. We found that NGFI-A mRNA is markedly up-regulated throughout the brains of animals exposed to the enriched environment, but not in the brains of either handled-only or undisturbed control groups. The most pronounced effects were observed in the somatosensory and visual cortices, in layers III and V, while more modest increases were observed in all other cortical layers, with the exception of layer I. A striking NGFI-A mRNA up-regulation was also observed in the striatum and hippocampal formation, notably in the CA1 subfield, of animals exposed to the enriched environment paradigm. Immunocytochemistry was also used to investigate the distribution of NGFI-A protein in response to the environmental enrichment protocol. A marked increase in the number of NGFI-A positive nuclei was identified in the enriched environment condition, as compared to undisturbed and handled-only controls, throughout the rat brain. While the greatest number of NGFI-A immunolabeled neurons was found in cortical layers III and V, up-regulation of NGFI-A protein was also detectable in layers II, IV and VI, in both the somatosensory and visual cortices. NGFI-A immunopositive neurons were also more numerous in the CA1 subfield of the hippocampal formation of animals exposed to the enriched environment, but remained at basal levels in both control groups. Our results implicate NGFI-A as one of the possible early genetic signals that ultimately lead to plastic changes in the CNS.

Induction of the 27-kDa heat shock protein (Hsp27) in the rat medulla oblongata after vagus nerve injury.

The 27-kDa heat shock protein (Hsp27) is constitutively expressed in motor and sensory neurons of the brainstem. Hsp27 is also rapidly induced in the nervous system following oxidative and cellular metabolic stress. In this study, we examined the distribution of Hsp27 in the rat medulla oblongata by means of immunohistochemistry after the vagus nerve was cut or crushed. After vagal injury, rats were allowed to survive for 6, 12, 24 h, 2, 4, 7, 10, 14, 30, or 90 days. Vagus nerve lesions resulted in a time-dependent up-regulation of Hsp27 in vagal motor and nodose ganglion sensory neurons that expressed Hsp27 constitutively and de novo induction in neurons that did not express Hsp27 constitutively. In the dorsal motor nucleus of the vagus nerve (DMV) and nucleus ambiguus, the levels of Hsp27 in motor neurons were elevated within 24 h of injury and persisted for up to 90 days. Vagal afferents to the nucleus of the tractus solitarius (NTS) and area postrema showed increases in Hsp27 levels within 4 days that were still present 90 days postinjury. In addition, increases in Hsp27 staining of axons in the NTS and DMV suggest that vagus nerve injury resulted in sprouting of afferent axons and spread into areas of the dorsal vagal complex not normally innervated by the vagus. Our observations are consistent with the possibility that Hsp27 plays a role in long-term survival of distinct subpopulations of injured vagal motor and sensory neurons.