Glial elements contribute to stress-induced torsinA expression in the CNS and peripheral nervous system.

DYT1 dystonia is caused by a single GAG deletion in exon 5 of TOR1A, the gene encoding torsinA, a putative chaperone protein. In this study, central and peripheral nervous system perturbations (transient forebrain ischemia and sciatic nerve transection, respectively) were used to examine the systems biology of torsinA in rats. After forebrain ischemia, quantitative real-time reverse transcriptase-polymerase chain reaction identified increased torsinA transcript levels in hippocampus, cerebral cortex, thalamus, striatum, and cerebellum at 24 h and 7 days. Expression declined toward sham values by 14 days in striatum, thalamus and cortex, and by 21 days in cerebellum and hippocampus. TorsinA transcripts were localized to dentate granule cells and pyramidal neurons in control hippocampus and were moderately elevated in these cell populations at 24 h after ischemia, after which CA1 expression was reduced, consistent with the loss of this vulnerable neuronal population. Increased in situ hybridization signal in CA1 stratum radiatum, stratum lacunosum-moleculare, and stratum oriens at 7 days after ischemia was correlated with the detection of torsinA immunoreactivity in interneurons and reactive astrocytes at 7 and 14 days. Sciatic nerve transection increased torsinA transcript levels between 24 h and 7 days in both ipsilateral and contralateral dorsal root ganglia (DRG). However, increased torsinA immunoreactivity was localized to both ganglion cells and satellite cells in ipsilateral DRG but was restricted to satellite cells contralaterally. These results suggest that torsinA participates in the response of neural tissue to central and peripheral insults and its sustained up-regulation indicates that torsinA may contribute to remodeling of neuronal circuitry. The striking induction of torsinA in astrocytes and satellite cells points to the potential involvement of glial elements in the pathobiology of DYT1 dystonia.

Peri-infarct depolarizations during focal ischemia in the awake Spontaneously Hypertensive Rat. Minimizing anesthesia confounds in experimental stroke.

Anesthesia profoundly impacts peri-infarct depolarizations (PIDs), but only one prior report has described their monitoring during experimental stroke in awake animals. Since temporal patterns of PID occurrence are model specific, the current study examined PID incidence during focal ischemia in the awake Spontaneously Hypertensive Rat (SHR), and documented the impact of both prior and concurrent isoflurane anesthesia. For awake recordings, electrodes were implanted under isoflurane anesthesia 1day to 5weeks prior to occlusion surgery. Rats were then subjected to permanent or transient (2h) tandem occlusion of the middle cerebral and ipsilateral common carotid arteries, followed by PID monitoring for up to 3days. Comparison perfusion imaging studies evaluated PID-associated hyperemic transients during permanent ischemia under anesthesia at varied intervals following prior isoflurane exposure. Prior anesthesia attenuated PID number at intervals up to 1week, establishing 2weeks as a practical recovery duration following surgical preparation to avoid isoflurane preconditioning effects. PIDs in awake SHR were limited to the first 4h after permanent occlusions. Maintaining anesthesia during this interval reduced PID number, and prolonged their occurrence through several hours following anesthesia termination. Although PID number otherwise correlated with infarct size, PID suppression by anesthesia was not protective in the absence of reperfusion. PIDs persisted up to 36h after transient occlusions. These results differ markedly from the one previous report of such monitoring in awake Sprague-Dawley rats, which found an extended biphasic PID time course during 24h after both permanent and transient filament occlusions. PID occurrence closely reflects the time course of infarct progression in the respective models, and may be more useful than absolute PID number as an index of ongoing pathology.

Evidence for the synthesis of tubulin on membrane-bound and free ribosomes from rat forebrain.

Evidence is presented that tubulin is synthesized in cell-free systems derived from rat forebrain containing either membrane-bound or free ribosomes. Both systems were supplemented with saturating amounts of messenger RNA (from either membrane-bound of free ribosomes) and soluble enzyme and initiation factors from whole forebrain. Synthesized products were chracterized by two biochemical purification methods for brain tubulin isolation. These methods of tubulin analysis indicated that both cell free systems synthesized the same relative amounts of tubulin.

Selective glial vulnerability following transient global ischemia in rat brain.

Global cerebral ischemia selectively damages neurons, but its contribution to glial cell death is uncertain. Accordingly, adult male rats were sacrificed by perfusion fixation at 1, 2, 3, 5, and 14 days following 10 minutes of global ischemia. This insult produces CA1 hippocampal neuronal death at post-ischemic (PI) day 3, but minor or no damage to neurons in other regions. In situ end labeling (ISEL) and immunohistochemistry identified fragmented DNA of dead or dying glia and distinguished glial subtypes. Rare ISEL-positive oligodendroglia, astrocytes, and microglia were present in control brain. Apoptotic bodies and ISEL-positive glia significantly increased at PI day 1 in cortex and thalamus (p < 0.05), but were similar to controls in other regions and at other PI intervals. Most were oligodendroglia, although ISEL-positive microglia and astrocytes were also observed. These results show that oligodendroglia die rapidly after brief global ischemia and are more sensitive than neurons in certain brain regions. Their selective vulnerability to ischemia may be responsible for the delayed white matter damage following anoxia or CO poisoning or that associated with white matter arteriopathies. Glial apoptosis could contribute to the DNA ladders of apoptotic oligonucleosomes that have been found in post-ischemic brain.

The heat shock/stress response in focal cerebral ischemia.

Focal ischemia results in striking changes in gene expression. Induction of hsp72, a member of the family of 70 kDa heat shock/stress proteins is a widely studied component of the generalized cellular response to injury known as the 'stress response' that is detected in brain after ischemia and other insults. This overview summarizes observations on hsp72 expression in models of focal cerebral ischemia, considering its cellular distribution, factors affecting its transcriptional and translational expression, and its potential relevance to post-ischemic pathophysiology. Hsp72 expression is essentially limited to regions in which cerebral blood flow falls below 50% of control levels, provided that residual perfusion allows synthesis of the induced mRNA and protein. The cellular distribution of hsp72 depends on the nature of the ischemic insult, with preferential vascular expression in severely ischemic territory that is destined to necrose, pronounced neuronal expression throughout the ischemic 'penumbra', and limited glial involvement in a narrow zone immediately surrounding the infarct. Together with results in other injury models, these observations indicate that hsp72 induction identifies discrete populations of surviving cells that are metabolically compromised, but not irreversibly damaged after focal ischemia. Available evidence suggests that the stress response is an important component of cellular defense mechanisms, and that successful accumulation of hsp72 is critical to survival following ischemia. Its expression may also contribute to mechanisms of induced ischemic tolerance. Future studies may be expected to more fully characterize the range of altered gene expression in response to focal ischemic injury and to establish specific roles for hsp72 and other induced proteins in the progression of injury and recovery following such insults.

Localization of 70 kDa stress protein mRNA induction in gerbil brain after ischemia.

Induction of mRNA encoding the 70 kDa stress/heat shock protein, hsp70, was evaluated in post-ischemic gerbil brain by in situ hybridization using an oligonucleotide probe selective for stress-inducible members of this gene family. Expression of hsp70 sequences was most pronounced in hippocampal CA1 neurons that fail to accumulate immunoreactive hsp70 protein, and that are selectively lost following ischemia. Hybridizable RNA continued to be expressed in CA1 through at least 48 h, essentially until the onset of cell death in this model. In contrast, dentate granule cells and CA2 neurons destined to survive the insult showed transient induction of hsp70 mRNA during the first 24 h of recirculation that disappeared prior to the detection of maximal hsp70 immunoreactivity in these cell populations. Pretreatment with a single injection of MK-801 (10 mg/kg) considerably attenuated the induction of hsp70 mRNA in hippocampus at 6 h of recirculation, an effect apparently mediated by persistent drug-induced hypothermia. The drug did not prevent the later, selective appearance of hsp70 hybridization in CA1 neurons at 24 h, nor did it protect against the subsequent loss of these cells. These results demonstrate a prolonged postischemic stress response at the transcriptional level in vulnerable hippocampal neurons, and suggest its utility as a marker for neuronal pathophysiology associated with mechanisms mediating delayed neuronal death.

Delayed increases in regional brain quinolinic acid follow transient ischemia in the gerbil.

Excessive activity or release of excitatory amino acids has been implicated in the neuronal injury that follows transient cerebral ischemia. To investigate the metabolism of the endogenous excitotoxin, quinolinic acid, and its potential for mediating cell loss following ischemia, the concentrations of quinolinic acid, L-tryptophan, 5-hydroxytryptamine, and 5-hydroxyindoleacetic acid were quantified in gerbil brain regions at different times after 5 or 15 min of ischemia induced by bilateral carotid artery occlusion. Significant elevation of brain tryptophan levels, accompanied by increased 5-hydroxyindoleacetic acid concentrations, occurred during the first several hours of recirculation, but regional brain quinolinic acid concentrations were found either to decrease or remain unchanged during the first 24 h after the ischemic insult. However, significant increases in quinolinic acid concentrations occurred in striatum and hippocampus at 2 days of recirculation after 5 min of ischemia. After a further 4 and 7 days, strikingly large increases in quinolinic acid concentrations were observed in all regions examined, with the highest levels observed in the hippocampus and striatum, regions that also show the most severe ischemic injury. These delayed increases in brain quinolinic acid concentrations are suggested to reflect the presence of activated macrophages, reactive astrocytes, and/or microglia in vulnerable regions during and subsequent to ischemic injury. While the results do not support a role for increased quinolinic acid concentrations in early excitotoxic neuronal damage, the role of the delayed increases in brain quinolinic acid in the progression of postischemic injury and its relevance to postischemic brain function remain to be established.

Reexpression of developmentally regulated MAP2c mRNA after ischemia: colocalization with hsp72 mRNA in vulnerable neurons.

Levels of mRNAs encoding the microtubule-associated proteins MAP2b and MAP2c as well as the 70-kDa stress protein [72-kDa heat shock protein (hsp72)] were evaluated in postischemic rat brain by in situ hybridization with oligonucleotide probes corresponding to the known rat sequences. Rats were subjected to 10-min cardiac arrest, produced by compression of major thoracic vessels, followed by resuscitation. The normally expressed MAP2b mRNA showed transient twofold elevations in all hippocampal neuron populations at 6-h recirculation, followed by a return to control levels by 24 h. MAP2b hybridization was progressively lost thereafter from the vulnerable CA1 and outer cortical layers, preceding both the fall in immunoreactive MAP2b and the eventual cell loss in these regions. The depletion of MAP2b mRNA coincided with an increase in the alternatively spliced MAP2c in vulnerable regions during 12-48 h of recirculation, precisely overlapping the late component of hsp72 expression that persisted in these cell populations. Previous studies have suggested that the initial induction of hsp72 provides an index of potential postischemic injury in neuron populations that may or may not be injured, while lasting hsp72 mRNA expression is associated with cell damage. In contrast, the present results demonstrate that MAP2c expression under these conditions occurs uniquely in neuron populations subject to injury. Available evidence suggests that MAP2c expression represents a plastic response in subpopulations of neurons that will survive in these regions, although it remains to be explicitly determined whether it may also be transiently expressed in dying cells. In any case, these observations demonstrate that reexpression of developmentally regulated MAP2c mRNA is a relatively late postischemic response in vulnerable cell populations, indicating that pathways regulating MAP2 splicing may be closely associated with mechanisms of neuron injury and/or recovery.

Cerebral vascular volume after repeated ischemic insults in the gerbil: comparison with changes in CBF and brain edema.

The time course of changes in cerebral intravascular volume was evaluated during 24 h following a series of three 5-min carotid artery occlusions spaced at 1-h intervals and compared with the changes occurring after single 5- or 15-min occlusions. Quantitative estimates of cerebral red cell volume, plasma volume, and total blood volume were obtained from the distribution spaces of 51Cr-labeled erythrocytes and 125I-albumin infused prior to killing at varied recirculation intervals. Significant reductions in vascular volume occurred in all ischemic brain regions within 1 h following a single 5-min occlusion, which recovered to control values within 6 h. A similar time course was seen after repeated occlusions. The reductions in volume remained significant at 6 h after a single 15-min occlusion, but there was no difference from control by 24 h. Thus, the time course of total vascular volume correlates well with that of CBF changes previously described, and both blood flow and blood volume are at normal levels during the time of severe edema 24 h after repeated occlusions. Calculated cerebral hematocrit was 60-70% of that obtained from the femoral artery, but was identical in all brain regions and was constant throughout the postischemic recirculation period, with the exception of a transient reduction in both peripheral and cerebral hematocrit observed at 6-h recirculation following single 15-min occlusions. These results suggest that changes in CBF and blood volume reflect primarily the status of larger vessels and that values in the normal range may be observed even under conditions of severe edema and impaired perfusion at the capillary level.

Effect of acute electrode placement on regional CBF in the gerbil: a comparison of blood flow measured by hydrogen clearance, [3H]nicotine, and [14C]iodoantipyrine techniques.

Regional cerebral blood flow (rCBF) was compared in the gerbil by means of [3H]nicotine, [14C]-iodoantipyrine, and hydrogen clearance techniques. In agreement with other studies, nicotine and iodoantipyrine methods gave virtually identical results. With these methods, it was observed that a reduction in blood flow occurred shortly after insertion of an electrode into the striatum for hydrogen clearance measurement, affecting rCBF throughout the impaled hemisphere. The reduction was moderate (30%) in the striatum and hippocampus, but much greater (70%) in cortical regions. Identical deficits were observed following brief penetrations involving only cortex. Following chronic electrode placement in the striatum, regional blood flow values obtained with [3H]nicotine returned to the control range within 6 h. Blood flow estimates obtained in the striatum with the implanted electrode increased with a similar time course, so that by 6-24 h, hydrogen clearance gave values indistinguishable from control values obtained with [3H]nicotine. These results clearly demonstrate that reduction of CBF subsequent to electrode placement can account for the low values frequently obtained with the hydrogen clearance method in small animals. The distribution of the deficit and the time course of its recovery are similar to blood flow changes associated with spreading depression. While mechanisms responsible for this effect remain to be fully identified, chronic implantation is a practical solution that allows the continued use of hydrogen clearance as a convenient method for repeated measurement of blood flow in the same animal.

Experimental model for repetitive ischemic attacks in the gerbil: the cumulative effect of repeated ischemic insults.

An experimental model for repeated ischemic attacks, which allows easy induction of cerebral ischemia of any desired duration and frequency, has been developed in the gerbil. With this procedure, a pronounced cumulative effect on development of edema and tissue injury was observed using 3 separate, 5-min bilateral occlusions of the common carotid arteries spaced at various time intervals. This effect was most evident when the occlusions were carried out at 1-h intervals, i.e., during the period of marked postischemic hypoperfusion. Such animals, killed after 24 h of recirculation, showed significantly more severe edema and brain tissue injury in the areas exposed to ischemia than was observed in animals killed 24 h after single 5- or 15-min occlusions. The changes of regional CBF, assayed with a [3H]nicotine method, indicated a relatively rapid onset of hypoperfusion of similar degree after each release of arterial occlusion. The hypoperfusion recovered significantly within 6 h of recirculation following either single or multiple occlusions, and no residual hypoperfusion was observed in animals which, when killed at 24 h, showed severe edema and brain tissue injury. This model should prove useful in elucidating the pathophysiological mechanisms operative in repetitive cerebral ischemia.

Heat-shock protein and C-fos expression in focal microvascular brain damage.

Cortical brain damage was produced in rats by a focal pulse from a Nd-YAG laser, and evolution of the lesion was evaluated at 30 min, and 2, 8, and 24 h with respect to microvascular perfusion, blood-brain barrier (BBB) permeability, and expression of both the heat-shock/stress protein, hsp72, and the c-fos proto-oncogene transcription factor. A double-labeling fluorescence technique employing intravenously injected Evans blue albumin (EBA) and fluorescein-labeled dextran was used to map and measure BBB damage and microvascular perfusion in fresh frozen brain sections. Hsp72 and c-fos mRNAs were localized by in situ hybridization, and the respective proteins were identified by immunocytochemistry. Parallel sections were stained for glial fibrillary acidic protein and for routine histologic examination. Striking hsp72 mRNA expression was evident by 2 h in an approximately 300 microns wide rim surrounding an area of expanding BBB damage. Increased hsp72 mRNA was observed only in regions of preserved microcirculation, where the hsp72 protein was subsequently localized exclusively in the vasculature at 24 h after the insult. Hsp72-positive endothelial cells spanned the narrow margin between the lesion and histologically normal, glial fibrillary acidic protein (GFAP)-positive cortical tissue. There was no hsp72 expression in the area of subcortically migrating edema fluid. Inductions of c-fos mRNA and Fos protein were not strikingly evident around the focal brain lesion, but were observed transiently throughout the injured hemisphere at 30 min and 2.5 h, respectively, indicating that spreading depression was triggered by the focal injury. These results are in striking contrast to those previously obtained from studies of models of focal ischemic or traumatic brain injury, which are characterized by a complex pattern of glial and neuronal hsp72 expression in the periphery of an infarct, and which suggest that the tightly demarcated lesion produced by the Nd-YAG laser lacks these components of graded injury that are evident following other types of focal brain damage.

Global cerebral ischemia associated with cardiac arrest in the rat: I. Dynamics of early neuronal changes.

Light microscopic neuronal changes were studied in rats subjected to 10 min of global ischemia produced by compression of the major cardiac vessels. Observations of cresyl violet-stained sections revealed early changes involving predominantly GABAergic neurons in various locations. In rats killed 15 min after recirculation, the changes were characterized by the appearance of a clear peripheral zone with condensation of the remaining neuronal cytoplasm. After 1 h, these zones appeared to be compartmentalized into individual pearl-like vacuoles, especially prominent in the nucleus reticularis thalami. After 3 h, the cytoplasmic vacuoles disappeared and the neuronal changes, particularly in the cerebral cortex, striatum, hippocampus, and pars reticulata of the substantia nigra, consisted mainly of hyperchromasia or loss of Nissl substance. After 2 days, the cerebral cortex and thalamus contained occasional neurons with conspicuously large nucleoli. After 7 days, the hippocampus revealed an approximately 50% loss of CA1 pyramidal neurons, associated with intense microglial reactivity in the stratum radiatum, whereas the neuronal destruction was more complete in the nucleus reticularis thalami. Our observations suggest a possibility that early changes in GABAergic neurons may provide a period of neuronal disinhibition and thus contribute to an excitatory ischemic damage in regions connected by GABAergic circuitry.

DNA fragmentation follows delayed neuronal death in CA1 neurons exposed to transient global ischemia in the rat.

Apoptosis is an active, gene-directed process of cell death in which early fragmentation of nuclear DNA precedes morphological changes in the nucleus and, later, in the cytoplasm. In ischemia, biochemical studies have detected oligonucleosomes of apoptosis whereas sequential morphological studies show changes consistent with necrosis rather than apoptosis. To resolve this apparent discrepancy, we subjected rats to 10 minutes of transient forebrain ischemia followed by 1 to 14 days of reperfusion. Parameters evaluated in the CA1 region of the hippocampus included morphology, in situ end labeling (ISEL) of fragmented DNA, and expression of p53. Neurons were indistinguishable from controls at postischemic day 1 but displayed cytoplasmic basophilia or focal condensations at day 2; some neurons were slightly swollen and a few appeared normal. In situ end labeling was absent. At days 3 and 5, approximately 40 to 60% of CA1 neurons had shrunken eosinophilic cytoplasm and pyknotic nuclei, but only half of these were ISEL. By day 14, many of the necrotic neurons had been removed by phagocytes; those remaining retained mild ISEL. Neither p53 protein nor mRNA were identified in control or postischemic brain by in situ hybridization with riboprobes or by northern blot analysis. These results show that DNA fragmentation occurs after the development of delayed neuronal death in CA1 neurons subjected to 10 minutes of global ischemia. They suggest that mechanisms other than apoptosis may mediate the irreversible changes in the CA1 neurons in this model.

Stress protein and proto-oncogene expression as indicators of neuronal pathophysiology after ischemia.

Induction of hsp70 mRNA and protein appear to provide useful markers for delineating stages in the progression of neuronal pathophysiology after ischemia. Detection of hsp70 encoded by the induced mRNA is dependent on complex interactions between the time course of mRNA expression and recovery of protein synthesis in a given neuron population, and perhaps other factors relating to specific aspects of hsp70 physiology, during recirculation intervals of hours to days. Transient mRNA expression and subsequent detection of immunoreactive hsp70 protein appear to identify neurons more likely to survive ischemia and other insults, while prolonged expression of hsp70 mRNA is associated with more severe neuronal injury. Fos and Jun immunoreactivities are also increased after ischemia, and provide indexes of functional gene expression during earlier recirculation periods. The accumulation of Fos immunoreactivity in particular designates neurons in which rapid recovery of protein synthesis during 1-3 h recirculation has allowed translation of the very transiently expressed c-fos mRNA. Jun-like immunoreactivity allows an evaluation of events at later recirculation intervals, and provides a clear demonstration of synthesis and accumulation of induced protein in CA1 neurons at 6 h following 2 min ischemia. Detailed understanding of the significance of such interactions between transcriptional and translational events will continue to evolve as information accumulates regarding the expression of additional mRNAs and proteins after ischemia. The present demonstration that Jun-like immunoreactivity accumulates in CA1 neurons after brief ischemia indicates that widespread changes in gene expression, expected as a consequence of such primary effects on transcription factor activity, are likely to contribute to the phenomenon of induced ischemic tolerance and to other persistent changes in the brain following diverse insults.

In vivo hyperthermia induces expression of HSP70 mRNA in brain regions controlling the neuroendocrine response to stress.

Regional localization of HSP70 expression in brain of rats exposed to increased ambient temperatures was examined using in situ hybridization. In addition to the cerebellar granule cell layer and choroid plexus, selective hybridization was observed in the hippocampal dentate gyrus, paraventricular and dorsomedial hypothalamic nuclei, median eminence, and medial habenula. Apparently, cells in brain regions coordinating the neuroendocrine response to stress show a preferential induction of cellular stress proteins in response to heat.

Coexpression of c-fos and hsp70 mRNAs in gerbil brain after ischemia: induction threshold, distribution and time course evaluated by in situ hybridization.

Levels of mRNAs encoding the proto-oncogene, c-fos, and the 70 kDa stress protein, hsp70, were evaluated in gerbil brain following transient cerebral ischemia of varied duration by in situ and blot hybridization techniques. Blots of total hippocampal RNA obtained after 5 min ischemic insults confirmed a characteristic, transient time course of c-fos expression with a striking elevation within 1 h and a return to control levels by 3 h recirculation. Hsp70 hybridization was significant at 1 h and continued to increase until 3-6 h after the insult. Striking accumulation of c-fos mRNA was detected within 15 min recirculation in dentate granule cells, persisting through 1 h, and a weaker signal was evident in CA1 and CA3 pyramidal neurons of hippocampus, as well as in prepiriform/entorhinal cortex and neocortical regions, during the same interval. Hsp70 hybridization showed an identical distribution at 1 h recirculation. Ischemic insults of 1 min duration resulted in no detectable increase of either mRNA, while 2 min ischemia resulted in changes comparable to those seen after 5 min insults. This common threshold corresponds to the ischemic interval required for energy depletion and resultant failure of intracellular ion homeostasis. In contrast, expression of hsp70 mRNA was not observed under conditions of brief depolarization accompanying cortical or hippocampal spreading depression that were shown to induce c-fos. A delayed component of c-fos mRNA expression was not detected in this model, while persistent hsp70 hybridization, restricted to hippocampal CA1 neurons, was evident at 48 h after either 2 min or 5 min ischemic insults. The parallels in c-fos and hsp70 mRNA expression during early recirculation suggest that overlapping mechanisms triggered following postischemic depolarization contribute to their induction after transient ischemia.

Synthesis of heat shock/stress proteins during cellular injury.

Several conclusions can be drawn from available data on the expression of stress proteins in brain with respect to their utility as markers of cellular injury. First, it is evident that all cell types in brain are capable of expressing stress proteins, although there is striking specificity in the population responding to a given insult. The apparent hierarchy of responsiveness indicated by hsp72 expression correlates well with the relative vulnerability of specific cell populations in a given model. With increasing severity of injury there can be an attenuation of the translational component of the stress response, in that hsp72 immunoreactivity fails to accumulate even though its mRNA is abundantly expressed. For this reason, hsp72 immunoreactivity provides an index of cell populations that have responded to an insult with a functional stress response. Such a response is not sufficient to guarantee survival, since many CA1 neurons that show significant hsp72 staining are eventually lost after global ischemia in the rat. However, brief insults that result in expression of hsp72 and other proteins encoded by induced mRNAs do result in tolerance to subsequent insults. Future studies may be expected to reveal the contributions of specific gene products to the tolerant state. Meanwhile, complementary evaluations of hsp72 mRNA and protein expression provide practical means of identifying cell populations responding to diverse injuries.

Distribution of phosphodiester and phosphorothioate oligonucleotides in rat brain after intraventricular and intrahippocampal administration determined by in situ hybridization.

The distribution and stability of exogenously administered oligonucleotides (oligos) are important variables determining the potential utility of antisense oligos as agents for modifying gene expression within a given brain region in vivo. In the present study, phosphodiester (PO) and phosphorothioate (PS) oligos antisense with respect to a recently cloned rat hsp70 sequence were localized in rat brain following intraventricular and intrahippocampal administration using an in situ hybridization detection method. Unlabeled PO and PS oligos were dissolved in artificial cerebrospinal fluid and infused under stereotaxic control using a syringe pump. At various intervals after administration frozen brain sections were collected on gelatin-coated slides and hybridized with 35S-labeled probe consisting of the corresponding phosphodiester sense sequence. After intraventricular administration the unmodified PO oligo exhibited a limited and strictly periventricular distribution. In contrast the PS oligo showed significant penetration into and accumulation within brain, with extensive uptake in ipsilateral striatum and dorsal hippocampus, as well as in midline periventricular structures. Both oligos remained detectable for at least two days after administration. Following intrahippocampal injection the PO oligo was rapidly lost from the injection site, with detectable signal persisting only along the hippocampal fissure at 24 h. The PS oligo exhibited a more diffuse initial distribution as well as greater stability. While there was no indication of specific accumulation in the major hippocampal neuron layers through 24 h, there was some indication of selective localization in neuronal soma by 48 h. These results confirm that the relative instability of unmodified oligos may severely limit their utility as antisense reagents in brain in vivo. While PS oligos show more widespread distribution than PO oligos after intraventricular infusion, even these do not detectably accumulate in cortex and other structures without immediate access to the ventricular space under the dosing conditions employed here. The hybridization approach used in these studies should prove to be of general use in verifying the targeting of specific brain structures with antisense oligos by various routes of administration.

Immunocytochemical and in situ hybridization approaches to the optimization of brain slice preparations.

Methods are described for determining the expression of specific mRNAs and proteins in brain slices, in order to elucidate changes in gene expression during preparation of vibratome slices from hippocampus of adult rats. In situ hybridization with 35S-labeled oligonucleotides was used to evaluate the level and distribution of c-fos and hsp72 mRNAs in 15-microns frozen sections prepared from these slices. Commercially available antibodies were used to examine the distribution of induced Fos and Jun proto-oncogenes as well as expression of the neuronal cytoskeletal protein, microtubule-associated protein 2 (MAP2), in 50-microns vibratome sections from immersion-fixed slices. These studies confirm the induction of c-fos and hsp72 mRNAs during routine incubation, as previously observed in hippocampal slices obtained with a tissue chopper and incubated under somewhat different conditions, indicating that such responses are likely to be common features of many slice preparations. Accumulation of Fos and Jun immunoreactivities in neurons and glia was generally consistent with the distribution of c-fos mRNA induction observed in slices, and the neuronal component of this response was comparable to the expression of these proteins observed after transient ischemia in vivo. MAP2 immunoreactivity detected in the dendritic processes of neurons tended to show an increase in staining intensity during slice incubation, although loss of dendritic staining in specific regions was occasionally observed in association with the absence of Fos and Jun expression and histological evidence of neuron damage. These results support the use of MAP2 immunoreactivity as a sensitive indicator of neuronal integrity in slices.(ABSTRACT TRUNCATED AT 250 WORDS)