Neuroscience Letters 282 (2000) 85±88 www.elsevier.com/locate/neulet
Transtentorial cerebellar c-jun expression after focal cerebral cortical injury in mice Jia-Shou Liu a, Yung-Yee Chang a, Hsiu-Shan Wu b, Chiung-Ying Huang c, Wei-Hsi Chen a, Min-Yu Lan a, Yi-Fen Kao a, Shun-Sheng Chen a,* a
Department of Neurology, Kaohsiung Medical University, 100 Shih-Chuan 1st Road, San-Ming District, Kaohsiung City 807, Taiwan b Department of Neurology, Kaohsiung Chang-Gung Memorial Hospital, Kaohsiung, Taiwan c Department of Neurosurgery, Program in Neuroscience, Stanford University School of Medicine, Palo Alto, California, CA, USA Received 5 January 2000; received in revised form 24 January 2000; accepted 25 January 2000
Abstract Delayed and remote effect of focal cerebral cortical lesion on cerebellum remains unclear. The c-Jun, an inducible transcription factor of cellular immediate early gene, is the predominant transcription factor and consistent marker for neurons that respond to stress or injury. We use a mouse cryogenic injury model to study the spatial and temporal changes of c-jun in the cerebellum after focal neocortical lesion. A transient and moderate expression of c-jun mRNA was found in the cerebellum with central dominance since 3 day postinjury and gradually subsided within 2 weeks. A distinct increment of c-Jun protein expression in Purkinje cells of the bilateral cerebellar hemispheres with focal connotation in the vermis was detected since 1 week postinjury. These ®ndings suggest that the delayed and remote c-jun expression of the cerebellum, functionally connected with the cerebral cortex, indicate transneuronal gene activation. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cryogenic cortical injury; c-jun; Cerebellum; Remote effect; Immunohistochemistry; In situ hybridization
Conventional biochemical and neuropathological studies on the central nervous system after focal cerebral injury have mainly focused on changes in the region of lesion or its surrounding areas. On the other hand, injury to a part of brain may cause neurochemical modi®cation or damage in areas distant from the primary site [18]. Similar observations had been documented in various models such as nigral degeneration secondary to striatal lesion [16,17], transhemispheric [11] or thalamic degeneration [7] following cortical infarction. Moreover, transneuronal degeneration via antegrade or retrograde pathway has been proposed as the cause of secondary neurodegeneration in remote areas following focal brain damage [7,11,16±18]. Diaschisis, the term was coined to describe the functional depression that occurs in neurons at distant areas of the brain following focal brain injury [6,19]. This phenomenon was initially conceptualized by von Monakow and he also proposed three types of diaschisis (diaschisis corticospinalis, diaschisis commissuralis, diaschisis associativa) as functional pathways of the * Corresponding author. Tel.: 1886-7-312-1101 ext. 6771; fax: 1886-7-311-2516. E-mail address:
[email protected] (S.-S. Chen)
neuropathological consequences [19]. Clinically, the socalled `crossed cerebro-cerebellar diaschisis' in patients with supratentorial stroke, tumor or head injury [6], has also been identi®ed by advanced imaging modalities, however, mechanisms underlying the contralateral cerebellar hypometabolism is still far from clear. The c-jun, a member of the immediate early gene family, is one of the consistent inducible markers for neurons that respond to stress or injury [9,12]. It also joins bipotential mechanisms that may lead to either degenerative or regenerative process of the nervous system [9]. In addition, c-Jun is a well-known transcription factor that expressed markedly in several brain injury models [9]. Its expression may also implicate cell damage or neuronal death in conditions such as traumatic brain injury, cerebral ischemia, hypoxia, seizure or degenerative disease [9,12]. To elucidate the complex cerebro-cerebellar effects after focal cortical injury, we examined the expression of c-jun and its protein in the cerebellum, as revealed by in situ hybridization and immunohistochemical method. In the present study, a clearly demarcated cortical lesion was made by well-established cryogenic brain injury model [3,13]. Focal cryogenic brain injury was carried out in accor-
0304-3940/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 0) 00 86 5- X
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dance with NIH guidelines and approved by the Animal Care Committee of the Kaohsiung Medical University. Three-month-old CD-1 male mice (35 ^ 5 g) were exposed to cryogenic injury of the right parietal cortex according to the method of Chan et al. [3]. Brie¯y the mice were anesthetized with xylazine (4 mg/kg) and ketamine (80 mg/kg) intraperitoneally and placed in a stereotactic apparatus for the following surgical procedures. After incising the scalp on the midline and removing the fascia, the skull was revealed. A metal probe, 4 mm in tip diameter pre-cooled with dry ice at 2808C, was applied in contact with the skull surface at 2 mm posterior to the Bregma and 2.0 mm lateral (right) to midline. Then the skull was exposed to a 30-s period of attachment with the probe by a weight of 100 g. This procedure can produce a reproducible focal full-layer cortical lesion localized to the right parietal cortex without affecting the underneath striatum and hippocampus. Shamoperated controls were subjected to the same procedure without using the chilled probe. Animals were sacri®ced at a predetermined post-injury interval, i.e. 1, 2, 4, 8 h, 1, 3, 7 and 14 days (n 3 per group) following cryogenic injury. The mice were overdosed by intraperitoneal chloral hydrate and were perfused via transcardic route with 100 ml of ten units heparin/ml physiological saline and 100 ml of 3.7% formaldehyde in 0.01 M phosphate-buffered saline (PBS). The brain were removed and post®xed in 3.7% formaldehyde/0.01 M PBS for 2 days then sliced to 20 or 50-mm-thick sections and prepared for the following experiments. In situ hybridization for c-jun mRNA was performed using the previously reported method [11] with modi®cation. Brain sections measuring 20 mm were mounted on SuperFrost Plus slides (Fisher Scienti®c) and subjected to 0.002% proteinase-K digestion at 378C for 15 min. The slides were washed in 0.1 M triethanolamine (TEA) and immersed in 0.25% acid anhydride/0.1 M TEA buffer for 10 min. A serial dehydration was performed and the slides were incubated with hybridization buffer containing 35Slabeled synthetic oligonucleotide (10 7 cpm/ml) at 428C overnight. The oligonucleotide probe of c-jun (48 mer) complementary to highly conserved amino acids 186±201 of the c-Jun protein coding sequence [15] was used as described earlier [10]. As a negative control, the sense probe was also employed in the experiment. After hybridization, the slices were washed in 1£ sodium chloride/ sodium citrate (SSC) at 558C for 120 min and serial dehydrated. The slides were exposed to Kodak BioMax MR-1 ®lm at room temperature (RT) for 2±4 weeks. The 50-mm-thick coronal free-¯oating sections were used for c-Jun immunohistochemical study. Before incubation the endogenous peroxidases were blocked with 0.66% H2O2 in 0.01M PBS with 0.3% Triton-X (T-PBS). After washed with T-PBS, the sections were incubated with 10% normal goat serum in T-PBS for 90 min at RT to decrease non-speci®c bindings. The slides were then incubated with a polyclonal rabbit anti-c-Jun IgG antibody (Santa Cruz Biotechnology) at
a dilution of 1:200 at 48C overnight. After rinsing with three changes of T-PBS, the slides were incubated with biotinylated goat anti-rabbit IgG secondary antibody (1:200 dilution; Vector Laboratories) at RT for 60 min. Subsequently the sections were stained by 0.02% 3,3 0 -diaminobenzidine hydrochloride (DAB) (Sigma) using Vectastatin ABC immunoperoxidase system (Vector Laboratories) and counterstained with 0.5% methylgreen, dehydrated and mounted. Negative controls were processed with normal rabbit IgG instead of primary anti-c-Jun antibody. Sections from each brain were also stained with cresyl violet for corresponding histological evaluation of brain damage. No animals died during or after cryogenic injury and no gross functional de®cit could be identi®ed. In agreement with previous reports [3,13], this model can cause a focal cortical lesion with a relatively distinct margin. In the experimental group, all the cerebellum subjected to histological examination did not exhibit neuronal damage (data not shown). The distribution of c-jun mRNA was assessed at predetermined intervals after cryogenic injury. On hybridized with sense probe, no visible expression could be found. However, there was a weak detectable c-jun mRNA in the brain, including cerebellum, at 0 h after treatment or shamoperated animals (Fig. 1A). Within one day postinjury, marked but transient c-jun mRNA was seen in the lesion hemisphere. Besides, moderate expressions were also found in the contralateral counterpart and subcortical structures, such as thalamus and striatum. Interestingly, a mild (Fig. 1B) to moderate (Fig. 1C) expression of c-jun mRNA was present in the bilateral cerebellar hemispheres with midline portion dominance since 1 to 3 days postinjury, respectively, and gradually subsided within 7 to 14 days survival (Fig.1D). When the primary antibody of c-Jun omitted from the
Fig. 1. In situ hybridization for c-jun mRNA (A±D). A weak detectable c-jun mRNA of the cerebellum was identi®ed in the shamoperated animals (A). In injured mice one day after injury (B), a mild increase of c-jun was noted; besides, mRNA levels were moderately elevated in the cerebellum with dominance in vermis at three days postlesion (C). The mRNA response in these injury-remote areas gradually subsided at 2 weeks postlesion (D).
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incubation, labeling was not detected in either sham-operated or injured brains. In sham-operated controls (Fig. 2A,B) or animals within 3 days postlesion, c-Jun immunoreactivity was detected in only a few cerebellar Purkinje cells. At 7 days after injury, distinct increment of c-Jun expression was found in the Purkinje cells of the bilateral cerebellar hemispheres symmetrically (Fig. 2D). Besides, distribution of the c-Jun immunoreactive cells showed focal connotation in the cerebellar vermis (Fig. 2C). Two weeks following the cryogenic injury, c-Jun immunoreactivity within the Purkinje cells still persisted. In the present study we demonstrate for the ®rst time that focal supratentorial cryogenic cortical injury induced transtentorial c-jun expression in the bilateral cerebellar hemispheres. The protein level of c-Jun, not constitutively expressed in the cerebellum, was detectable as early as 7 days and up to 14 days after injury. And this manifestation was preceded by the expression of c-jun mRNA 3 days after injury. This speci®c temporal relationship suggests that cJun protein expression was attributable to the increased cjun mRNA in areas identi®ed by in situ hybridization. As compared with our previous observation of the transhemispheric c-fos activation in a focal cortical ischemia-reperfusion model [11], the transtentorial c-jun expression in the present study involved a more protracted course. The product of c-jun gene, an important nuclear regulatory element, responds promptly to a series of stimuli [12]. As known it acts independently or in combination with c-Fos that bind to the AP-1site regulating downstream gene expressions. Several studies have shown that in neurons
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likely to die, c-Jun expression was induced for a long period while c-Fos or Fos-B was transient in nature [5,9]. Using the same injury model, our preliminary results also show that cJun expression is the predominant protein in the AP-1 complexes shown by gel mobility shift assay [unpublisheddata]. In developing neurons, c-jun expression apparently plays an important role in the programmed cell death [9]. In addition, c-jun induction has been related to diverse nervous system disorders, such as cerebral ischemia, trauma, ionizing radiation, Alzheimer's and other degenerative disease [9]. The above ®ndings may support the pivotal role of c-jun on the neuronal fate. This cryogenic cortical injury model has been considered not only as a representation of vasogenic brain edema characterized by increased blood±brain barrier (BBB) permeability, but also an ideal one for the study of secondary brain injury in which necrosis and apoptosis play important roles [13]. It is inappropriate to describe this delayed and distant gene expression to the direct focal injury or brain edema, although a mild BBB damage could be identi®ed in this model within 3 days postinjury [4,13]. Using a forebrain lateral ¯uid percussive impact model, Fukuda et al. [8] demonstrated similar phenomenon that the Purkinje cells were vulnerable to the mild injury, however, the putative mechanism of Purkinje cell death remained unknown. In the present study, c-jun mRNA as well as c-Jun protein expression migrated from the cerebral cortex and subcortical structures to the cerebellum. It is important to point out that c-jun and c-Jun expression in the Purkinje cells is primarily located in the parasagittal zone of the cerebellum, especially
Fig. 2. Immunohistochemistry of c-Jun. (A±D, 100£). In sham-operated mice, scattered immunoreactive cells were observed in the Purkinje of the vermis (A) or the cerebellar hemisphere (B). One week after cryogenic injury, marked increase of the immunoreactive Purkinje cells (arrow heads) in the remote areas of the vermis (C) or the cerebellar hemisphere (D) were identi®ed.
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the vermis. Neuroanatomically, this is in accordance with the speci®c topographic organization of olivocerebellar climbing ®ber projection [20]. These ®ndings denote that certain transneuronal pathways were activated in response to the focal cortical insult. Of special note, interruption of the cerebro-ponto-cerebellar loop with subsequent loss of the inhibitory signaling is the most likely mechanism of this distant transneuronal gene expression [6]. Following injury, disinhibited inputs through excitatory amino acid receptors of the Purkinje cells via the climbing and parallel ®ber may underlie the basis of this remote gene response [14]. In other words, the excitotoxicity paradigm may orchestrate phenotypic changes in the cerebellum remote from the injury site. Aside from this, several studies have demonstrated delayed and remote gene expression after focal cerebral ischemia, especially in the homotopic area of the contralateral hemisphere [1,2]. Additionally, this remote alteration might also lead to widespread postlesional cytoskeletal changes in the functionally de®ned brain region [1]. In conclusion, we proposed that the delayed and remote c-jun expression in the cerebellum as an applicable marker of disordered transneuronal communications. However, more studies are needed to clarify its relationship with the possible secondary Purkinje cell degeneration following supratentorial injury. The authors are grateful to C.J. Pang, for her excellent technical assistance. This work was supported by the Taiwan National Science Council grants: NSC-88±2314B-037±076, NSC-89±2314-B-037±037, NSC-89±2314-B037±039. [1] Bidmon, H.J., Jancsik, V., Schleicher, A., Hagemann, G., Witte, O.W., Woodhams, P. and Zilles, K., Structural alternations and changes in cytoskeletal proteins and proteoglycans after focal cortical ischemia. Neuroscience, 82 (1997) 397±420. [2] Bidmon, H.J., Kato, K., Schleicher, A., Witte, O.W. and Zilles, K., Transient increase of manganese-superoxide dismutase in remote brain areas after focal photothrombotic cortical lesion. Stroke, 29 (1998) 203±211. [3] Chan, P.H., Yang, G.Y., Chen, S.F., Carlson, E. and Epstein, C.J., Cold-induced brain edema and infarction are reduced in transgenic mice overexpressing CuZn-superoxide dismutase. Ann. Neurol., 29 (1991) 482±486. [4] Chang, Y.Y., Huang, C.Y., Morita-Fujimura, Y., Copin, J.C. and Chan, P.H., Widespread nuclear factor-kappa B activation in brain after focal cortical cold injury. J. Neurotrauma, 15 (1998) 862. [5] Dragunow, M., Young, D., Hughes, P., MacGibbon, G., Lawlor, P., Singleton, K., Sirimanne, E., Beilharz, E. and Gluckman, P., Is c-jun involved in nerve cell death following
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