Genetic influences on cellular reactions to CNS injury: The reactive response of astrocytes in denervated neuropil regions in mice carrying a mutation (WldS) that causes delayed Wallerian degeneration

This study compares the reactive changes in astrocytes in denervated neuropil regions in normal mice and in mice carrying the Wld^S mutation which leads to delayed Wallerian degeneration. In situ hybridization and immunocytochemical techniques were used to define the time course of changes in the levels of glial fibrillary acidic protein (GFAP) and GFAP mRNA in the denervated neuropil of the hippocampus after unilateral aspiration lesions of the entorhinal cortex. In control mice, GFAP mRNA levels increased rapidly in the denervated neuropil to a peak that was about tenfold higher than control at 2–4 days, decreased between 6 and 8 days postlesion, and then increased again to a second peak at 10 days postlesion. Increases in immunostaining for GFAP were evident by 2 days, remained elevated until 12 days postlesion and then decreased slowly. In mice carrying the Wld^S mutation, the upregulation of GFAP mRNA levels in the denervated laminae was substantially delayed. Strikingly absent was the dramatic increase in labeling at 2–4 days postlesion which was such a prominent feature of the response in control animals. Peak labeling in the denervated laminae was not seen until 10–12 days postlesion. The development of a well-defined band of intensely immunostained and hypertrophied astrocytes in the denervated zone was also delayed in the Wld^S animals, although there were modest increases in immunostaining as early as 2 days postlesion that were seen throughout the hippocampus ipsilateral to the lesion. These results suggest that degenerative changes in axons and synaptic terminals are the principal trigger for upregulating GFAP expression in the denervated neuropil, although other signals also play a role in the early postlesion response. J. Comp. Neurol. 380:70–81, 1997. © 1997 Wiley-Liss, Inc.     

Laser microdissection of immunolabeled astrocytes allows quantification of astrocytic gene expression

Astrocytes represent the major glial cell population within the central nervous system. In order to elucidate the function of astrocytes under physiological conditions and during the course of neurological disease, astrocytic gene expression profiling is necessary. However, since astrocytes form an intimately connected network with neurons and other cell types in the brain, gene expression analysis of astrocytes with a sufficient degree of cellular specificity is difficult. Here we are presenting a rapid and, thus, RNA preserving immunostaining protocol for the detection of astrocytes in rodent brain. This protocol can readily be combined with laser microdissection (Leica AS LMD platform) and quantitative RT-PCR (qPCR). Employing this method, we studied changes in glial fibrillary acidic protein (GFAP) expression in astrocytes of mouse entorhinal cortex following entorhinal cortex lesion. Using laser microdissection, astrocytes (n = 60) were collected in the tissue surrounding the lesion, the entorhinal cortex contralateral to the lesion, and in unlesioned control animals. Changes in GFAP mRNA were quantified using qPCR. GFAP mRNA levels were 82-fold higher in astrocytes of lesioned animals at the site of the lesion compared to GFAP mRNA levels in entorhinal cortex astrocytes of control mice. GFAP mRNA levels were only slightly elevated at the contralateral side (lesioned animals). This optimized protocol for immunolabeling and laser microdissection of astrocytes followed by qPCR allows quantification of astrocytic gene expression levels with a high degree of cellular specificity. It may similarly be employed in different settings where other cell types need to be identified and microdissected for gene expression profiling.     

Entorhinal deafferentation induces upregulation of SPARC in the mouse hippocampus

SPARC is a matricellular protein that modulates cell-cell and cell-matrix interactions by virtue of its antiproliferative and counteradhesive properties. Here, we report the denervation-induced upregulation of SPARC mRNA and protein in the mouse hippocampus following transections of the entorhinal afferents. Northern blot analysis showed that SPARC mRNA was upregulated in a transient manner in the deafferented mouse hippocampus. In situ hybridization and immunohistochemistry confirmed the temporal upregulation of both SPARC mRNA and protein specifically in the denervated areas, which initiated at 7 days postlesion, reached the maximum at 15 as well as 30 days postlesion, and subsided towards normal levels by 60 days postlesion. Double labeling by either a combination of in situ hybridization for SPARC mRNA with immunohistochemistry for glial fibrillary acidic protein or double immunofluorescence staining for both proteins in the hippocampus revealed that SPARC-expressing cells are reactive astrocytes. In respect to the spatiotemporal alterations of SPARC expression in the denervated hippocampus, we suggest that SPARC may be involved in modulation of the denervation-induced plasticity processes such as glial cell proliferation, axonal sprouting and subsequent synaptogenesis in the hippocampus following entorhinal deafferentation.     

Increases in transforming growth factor-beta mRNA in hippocampus during response to entorhinal cortex lesions in intact and adrenalectomized rats

Transforming growth factor-beta mRNA was detected with a rat TGF-beta 1 coding sequence probe as a 2.5 kb band by RNA blot hybridization of total RNA from the adult male rat hippocampus. Following electrolytic lesions of the entorhinal cortex that cause hippocampal deafferentation and synaptic remodeling, TGF-beta mRNA increases 5-fold in ipsilateral hippocampus when compared with intact controls. This increase was independent of prior adrenalectomy or corticosterone-replacement. These data demonstrate that TGF-beta gene expression increases in response to hippocampal deafferentation.     

Corticosterone differentially regulates the bilateral response of astrocyte mRNAs in the hippocampus to entorhinal cortex lesions in male rats.

This study examined the effect of adrenalectomy (ADX) and corticosterone (CORT) replacement on the levels of two astrocyte mRNAs during responses to unilateral entorhinal cortex lesions (ECL) to identify molecular mechanisms involved in glucocorticoid modulation of astrocyte activation following deafferentation. Both glial fibrillary acidic protein (GFAP) and sulfated glycoprotein-2 (SGP-2) mRNA were increased in the ipsilateral hippocampus 4 days following unilateral ECL. In unlesioned ADX rats CORT replacement decreased both messages in the hippocampus. CORT replacement suppressed the ECL-induced increase of GFAP mRNA in the contralateral, but not ipsilateral hippocampus of ADX rats. In contrast, CORT decreased SGP-2 mRNA both ipsi- and contralaterally. It is clear that several regulatory mechanisms are responsible for maintaining a physiological balance of astrocyte activity in the adult brain, and that changes in circuit integrity and the endocrine milieu can alter this balance.     

Effect of a discrete dorsal forebrain lesion in the rat on the expression of neuronal and glial-specific genes: induction of calmodulin, NF-L, SC1, and GFAP mRNA.

The influence of a localized tissue injury on the expression of genes encoding specific neuronal and glial proteins was examined using in situ hybridization. A pronounced induction of neuron-specific calmodulin (CaM) mRNA was evident within cells proximal to the wound site by 2 hours following a cortical lesion in rats. By 12 hours postlesion, intense signal corresponding to CaM mRNA was found to extend 1 mm from the wound site. Changes in the expression of mRNA encoding two additional neuronal proteins, the 68 kilodalton neurofilament protein and the extracellular matrix protein, SC1, were also evident at 12 hours following the cortical injury. Of the two glial proteins examined, a dramatic elevation in levels of mRNA for glial fibrillary acidic protein (GFAP) was observed at the wound site by 12 hours postlesion. This intense labeling corresponding to GFAP mRNA was evident in the ipsilateral glial limitans and hippocampus as well as in the contralateral glial limitans. In contrast, the pattern of labeling for the beta-subunit of the S100 protein did not differ from that of control animals at either of the postlesion intervals examined. This study identifies four genes, CaM, GFAP, SC1, and NF-L, which are induced following a localized cortical injury and which encode mRNA species enriched in specific cell-types in the central nervous system.     

Kainate receptors in the rat hippocampus: a distribution and time course of changes in response to unilateral lesions of the entorhinal cortex

The response of kainate receptors to deafferentation and subsequent reinnervation following unilateral entorhinal cortex lesions was studied in the rat hippocampus using quantitative in vitro autoradiography. The binding levels of [3H]kainic acid (KA) and changes in the distribution of KA sites were investigated in the dentate gyrus molecular layer (ML) and in various terminal zones in the CA1 field at 1, 3, 7, 14, 21, 30, and 60 d postlesion. The data from both the ipsilateral and contralateral hippocampus were compared with those from unoperated controls. The first changes in KA receptor distribution were observed 21 d postlesion when the dense band of KA receptors occupying the inner one-third of the ML expanded into the denervated outer two-thirds of the ipsilateral ML. The spreading of the KA receptor field into previously unoccupied zones continued 30 and 60 d postlesion. At these time points, the zone enriched in [3H]KA binding sites became significantly (on average 50%) wider than in unoperated controls. No changes were observed in either the distribution or binding levels in other hippocampal areas or in the contralateral hippocampus at any studied time point. Saturation analysis of binding in the ipsilateral ML 60 d postlesion revealed changes in the maximum number of receptor sites (Bmax) without changes in KA receptor affinity (Kd). The data suggest that the elevation of the [3H]KA binding in the outer two-thirds of the ML reflects an increase in the number of both low and high affinity receptor binding sites. The pattern of KA receptor redistribution was similar to the well-characterized pattern of sprouting of commissural/associational systems from the inner one-third into the outer two-thirds of the ML after entorhinal lesions (Zimmer, 1973; Lynch et al., 1975). This supports the hypothesis (Geddes et al., 1985) that the KA receptor response observed in the present study reflects postlesion reorganization of inputs within the denervated ML and may be relevant to functional recovery of the damaged circuits.     

Altered expression of microtubule-associated protein 1B in cerebral cortical structures of pentylenetetrazole-treated rats

Using Northern blot, immunoblotting, immunocytochemistry, and in situ hybridization, we show that a single administration of the convulsant pentylenetetrazole leads to robust, long-term changes in microtubule-associated protein 1B and its mRNA, in the adult rat brain. The first increases in MAP1B mRNA were detected at 15 hr following pentylenetetrazole administration in the temporal (Te2) and perirhinal cortex followed by increases in microtubule-associated protein 1B immunoreactivity at 72 hr postseizure. In contrast, the levels of microtubule-associated protein 1B mRNA and protein in layers I–II of the retrosplenial and parietal cortex (Par2) declined visibly by 24 hr and 72 h, respectively, post-seizure. The changes included loss of staining in layers I–II and development of structures resembling “strings-of-beads” along the fibers of projection neurons of layer V. The levels of microtubule-associated protein 1B mRNA in the entorhinal cortex peaked at later times (72 h), especially in layers II–III, and returned to control levels by 10 days. Whereas the levels of microtubule-associated protein 1B immunoreactivity in the retrosplenial and parietal cortex recovered by 5–10 days, it persisted at high levels through day 35 in layer V of the temporal cortex (Te2), layers II–III of the perirhinal cortex and layers I–II of the lateral entorhinal cortex. These results indicate that seizure activity leads to long-term upregulation of genes coding for structural elements that are characteristic of the immature brain such as microtubule-associated protein 1B. J. Neurosci. Res. 51:646–657, 1998. © 1998 Wiley-Liss, Inc.     

Long-term astroglial reaction to serotonergic fiber degeneration

Glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS) expression were analysed by Western and Northern blotting in the hippocampus, the frontal and occipital cortex, and the cerebellum of the adult rat, as a manifestation of the astroglial reaction, 2 and 3 months after 5,7-dihydroxytryptamine injection into the lateral ventricule. 5HT injury stimulated GFAP and GS expression in a temporally and regionally specific fashion. At 2 months postlesion, the GFAP-mRNA and GFAP levels appeared enhanced but returned to control levels at 3 months. The GFAP-mRNA and GS-mRNA levels increased in the frontal cortex at 3 months. Such a delayed astroglial reactivity might implicate astrocytes in neurodegenerative disorders.     

Enkephalin biosynthesis and enkephalin gene expression are increased in hippocampal mossy fibers following a unilateral lesion of the hilus

The biosynthesis and posttranslational processing of proenkephalin and the level of preproenkephalin mRNA were investigated in the mossy fiber system of the granule cells of the hippocampus in the presence or absence of a unilateral lesion of the hilus, a procedure that produces an episode of recurrent bilateral hippocampal seizures lasting several hours. Both immunocytochemistry and radioimmunoassay (RIA) have demonstrated that the hilus lesion leads to large bilateral increases in enkephalin immunoreactivity in the mossy fiber system. In the present study, RIA data indicate that following an initial decline in immunoreactivity, enkephalin content within the mossy fibers first begins to increase between 18 and 24 hr after lesioning. Using the technique of in vivo radiolabeling and high-performance liquid chromatographic purification of identified radiolabeled peptides, we observed a 14-fold increase in incorporation of radiolabeled methionine into Met5-enkephalin at 24-30 hr postlesion, as compared with control animals, when Met5-enkephalin was purified from the mossy fiber terminal fields. To examine the posttranslational proteolytic processing of proenkephalin, the biosynthesis of 5 additional Met5-enkephalin-containing peptides was also examined. We determined that the posttranslational processing of proenkephalin did not yield exclusively penta-, hepta-, and octapeptides but larger opioid peptides as well in both control and lesioned animals, and that the ratio of the enkephalin peptides was not altered following the lesion. Measurement of preproenkephalin messenger RNA levels in the granule cells by Northern analysis revealed a marked increase following the lesion. Compared with the control animals, preproenkephalin mRNA was 8.5-fold higher in the contralateral dentate gyrus at 12 hr postlesion and 14- to 15-fold higher by 24 hr.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transient increases and individual variations in preprocholecystokinin gene expression in rat telencephalic regions during postnatal development: a quantitative comparison between preprocholecystokinin mRNA and preproenkephalin mRNA

Postnatal development of the tissue levels of mRNAs encoding preprocholecystokinin and preproenkephalin in rat telencephalic regions was analyzed by quantitative dot blot hybridization using synthetic RNAs as standards. Preprocholecystokinin mRNA levels per tissue weight showed distinct peaks in the frontal cortex and hippocampus on postnatal days 10 and 20, respectively, while preproenkephalin mRNA levels in these regions showed no apparent peaks. In the striatum, preproenkephalin mRNA abundance linearly increased 8-fold from day 5-43, while a transient increase in the preprocholecystokinin mRNA abundance between days 10 and 20 was noted. Furthermore, the striatal levels of preprocholecystokinin mRNA showed marked individual variations during the early period of postnatal development. These results suggest transient and variable increases in cell density and/or in gene expression of cholecystokinin-synthesizing cells in the developing telencephalon.     

Astrocytic apolipoprotein E mRNA and GFAP mRNA in hippocampus after entorhinal cortex lesioning.

Entorhinal cortex lesions (ECL) that damage the perforant path to the hippocampus induce rapid increases of apolipoprotein E (apo E) mRNA in the hippocampus. Apo E mRNA was localized in astrocytes by in situ hybridization in combination with immunocytochemistry for glial fibrillary acidic protein (GFAP). Unilateral ECL also increased hippocampal GFAP mRNA, with increases preceding those of apo E mRNA. The apo E mRNA and GFAP mRNA responses were transiently bilateral in non-denervated zones. The timing of response in apo E mRNA to deafferentation supports suggestions that apo E has roles in membrane remodelling during responses to neuron injury.     

Kainic acid alters the metabolism of Met5-enkephalin and the level of dynorphin A in the rat hippocampus

Male Fischer-344 rats were given a single intrastriatal injection of kainic acid (KA; 1 microgram/rat), which caused recurrent motor seizures lasting 3-6 hr. During the convulsive period, native Met5-enkephalin-like (ME-LI) and dynorphin A (1-8)-like (DYN-LI) immunoreactivities in hippocampus decreased by 31 and 63%, respectively. By 24 hr after dosing, the hippocampal opioid peptides had returned to control levels, and by 48 hr ME-LI had increased 270% and DYN-LI 150%. Immunocytochemical analysis revealed that ME-LI and Leu5-enkephalin-like (LE-LI) immunostaining in the mossy fibers of dentate granule cells and the perforant-temporoammonic pathway had decreased visibly by 6 hr and had increased markedly by 48 hr following KA. A visible decrease in DYN-LI in mossy fiber axons within 6 hr was followed by a substantial increase by 48 hr. To determine whether the increases in hippocampal ME-LI reflected changes in ME biosynthesis, levels of mRNA coding for preproenkephalin (mRNAenk) and cryptic ME-LI cleaved by enzyme digestion from preproenkephalin were measured. Following the convulsive period (6 hr), mRNAenk was 400% of control, and by 24 hr, cryptic ME-LI was 300% of control. Increases in native and cryptic ME-LI and in mRNAenk were also noted in entorhinal cortex, but not in hypothalamus or uninjected striatum. Our data suggest that KA-induced seizures cause an increase in ME release, followed by a compensatory increase in ME biosynthesis in the hippocampus and entorhinal cortex.     

Transient upregulation of NCAM mRNA in astrocytes in response to entorhinal cortex lesions and ischemia

Axonal sprouting and synaptic reorganization play an important role in the adaptation of the CNS to injury. However, the molecular mechanisms underlying this neuronal plasticity are poorly understood. In the present study we used in situ hybridization to examine the expression of NCAM mRNA in normal hippocampus, and in response to entorhinal cortex (EC) lesions and transient global ischemia. Both neurons and astrocytes were labeled by digoxygenin-tagged cRNA probes which recognize all three major NCAM isoforms of the adult CNS. In contrast, NCAM180-specific probes labeled only neurons in the hippocampus. After unilateral EC lesion, a transient and anatomically restricted upregulation of NCAM120/140 mRNA in reactive astrocytes in the denervated molecular layer of the dentate gyrus was observed. This increase was only present 2–4 days after the lesion whereas the GFAP mRNA increase was present up to 30 days postlesion. Following global ischemia a similar, transient increase of NCAM120/140 mRNA labeling of reactive astrocytes was observed; this increase was anatomically restricted to CA1, where neuronal loss occurred. Results suggest that the transient upregulation of NCAM120/140 mRNA in reactive astrocytes shortly after injury might be an important molecular mechanism in the cascade of events underlying neuronal plasticity in the adult CNS.     

Insulin-like growth factor-1 mRNA is increased in deafferented hippocampus: Spatiotemporal correspondence of a trophic event with axon sprouting

Deafferentation is known to induce axonal sprouting in adult brain, but the signals that direct this response are not understood. To evaluate the possible roles of insulin-like growth factor-1 (IGF-1) and basic fibroblast growth factor (bFGF) in central axonal sprouting, the present study used in situ hybridization to evaluate IGF-1 and bFGF mRNA expression in entorhinal deafferented rat hippocampus. Alternate tissue sections were processed for Fink-Heimer impregnation of axonal degeneration, Bandeiraea simplicifolia (BS-1) labeling of microglia, and glial fibrillary acidic protein immunocytochemistry. In control hippocampus, IGF-1 mRNA was localized to a few neurons, with no labeled cells in the dentate gyrus molecular layer; bFGF cRNA hybridization was diffuse in dendritic fields but was dense in CA2 stratum pyramidale. Both mRNA species were increased by deafferentation. The distribution of elevated IGF-1 mRNA corresponded precisely to fields of axonal degeneration and was greatest in the dentate gyrus outer molecular layer and stratum lacunosum moleculare. In these fields, IGF-1 mRNA was elevated by 2 days, reached maximal levels at 4 days, and declined by 10 days postlesion. Double labeling revealed that the majority of IGF-1 cRNA-labeled cells were microglia. In deafferented hippocampus, bFGF mRNA was broadly increased across fields both containing and lacking axonal degeneration. In the dentate, bFGF mRNA levels peaked at 5 days postlesion and remained elevated through 14 days. These results demonstrate that reactive microglia within deafferented hippocampal. Laminae express IGF-1 mRNA just prior to and during the period of reactive axonal growth and suggest that IGF-1 plays a role in directing the sprouting of spared afferents into these fields. © 1995 Wiley-Liss, Inc.     

Transient ischemia stimulates glial fibrillary acid protein and vimentin gene expression in the gerbil neocortex, striatum and hippocampus.

Astrocytic activation plays a major role in homeostatic maintenance of the central nervous system in response to neuronal damage. To assess the reactivity of astrocytes in transient cerebral ischemia of the gerbil, we studied the levels of glial fibrillary acidic protein (GFAP) and its mRNA. GFAP mRNA increased by 4 h after carotid artery occlusion, reached peak levels by 72 h with a 12-fold increase over control and then started declining as early as 96 h postischemia. An examination of the specific regions of the brain revealed an increase in GFAP mRNA associated with the forebrain, midbrain, hippocampus and striatum. GFAP mRNA in the non-ischemic cerebellum however, remained expressed at constitutively low levels. Immunoblot analysis with anti-GFAP antibodies demonstrated a 2- to 3-fold increase in the protein after 24 and 48 h of reperfusion. Pretreatment with pentobarbital and 1-(5'-oxohexyl)-3-methyl-7-propyl xanthine (HWA 285), the drugs that have been shown to protect against ischemic damage, prevented the increase in GFAP mRNA in the cortex following ischemic injury. Forebrain ischemia also induced vimentin mRNA and protein quantities by 12 h of reperfusion in the cortex. The levels of c-fos and preproenkephalin mRNA increased rapidly within 1 h after ischemic injury, demonstrating a temporal difference in mRNA changes following ischemia. These results indicate that an increase in GFAP and vimentin, the two glial intermediate filament proteins in the area of the ischemic lesion may be associated with a glial response to injury.