Neurofilament expression in the rat brain after cerebral infarction: effect of age

Neurobiology of Aging 24 (2003) 135–145

Neurofilament expression in the rat brain after cerebral infarction: effect of age E. Schroeder a , S. Vogelgesang a , A. Popa-Wagner b , C. Kessler b,∗ a

Department of Pathology, Ernst-Moritz-Arndt-University, Friedrich-Loeffler-Strasse 23e, Greifswald 17487, Germany b Department of Neurology, Ernst-Moritz-Arndt-University, Ellernholzstrasse 1-2, Greifswald 17487, Germany Received 2 October 2001; received in revised form 22 February 2002; accepted 2 April 2002

Abstract In this study the role of neurofilaments (NFs) in brain plasticity after cerebral infarction in young and middle aged rats was evaluated. Focal cerebral ischemia was produced by reversible occlusion of the right middle cerebral artery in 3- and 20-month-old male Sprague–Dawley rats. After 1 week, brains were removed and in situ hybridization and immunostaining was performed for NF-68 kDa, 160 kDa and 200 kDa in different phosphorylation states. After focal cerebral ischemia the levels of gene and protein expression of neurofilament proteins were increased in the border zone of the infarcted area compared with the unaffected contralateral site. Furthermore, the level of gene expression was significant lower in aged as in young animals. Focal cerebral ischemia resulted in a clearly increased number of immunostained axons in the penumbral region in both young and aged rats. On the other hand the immunostained apical dendrites became thicker and vacuolization appeared. Our results suggest that that neurofilament proteins are involved in response of brain to focal ischemia. © 2002 Published by Elsevier Science Inc. Keywords: Neurofilament proteins; Stroke; Brain plasticity; Aging; Rat

1. Introduction Brain injury leads to neuronal degeneration with loss of synaptic connectivity. In response, compensatory axonal sprouting in the surviving deafferented neurons [6] leads to the establishment of new synaptic contacts with other nerve cells [12]. In this context, cytoskeletal elements such as neurofilament proteins play an important role in the response of the brain to damage. Neurofilaments (NFs) are neuron-specific intermediate filaments that are composed of three major subunits designated NF-L (68 kDa), NF-M (160 kDa) and NF-H (200 kDa). These neurofilament proteins are involved in multiple plasticity processes; NF-68 is expressed in axonal growth cones [13]. NF-M and NF-H are involved in the stabilization of newly-sprouted axonal processes [20,28] while NF-H is thought to play an important role primarily in the stabilization and maturation of pre-existing connections [14,16]. Neurofilament proteins, especially NF-160 and NF-200, are among the most phosphorylated proteins in the brain. Maybe the extent and localization of phosphorylation are involved in the distribution and biological functions of neurofilaments in neurons [8,15]. ∗

Corresponding author. Tel.: +49-3834-866815; fax: +49-3834-866875. E-mail address: [email protected] (C. Kessler).

0197-4580/02/$ – see front matter © 2002 Published by Elsevier Science Inc. PII: S 0 1 9 7 - 4 5 8 0 ( 0 2 ) 0 0 0 6 3 - 5

The aim of the present study was to investigate the involvement of different phosphorylated NFs in the cerebral response to stroke in young and aged rats. We focused especially on the border zone, or penumbra, of the infarct because the persistence of viable neuronal elements in this region makes it an important site for potential therapeutic interventions. If protection is not achieved in the penumbra, the neurons there will die and become part of the infarct core [24]. Most experimental studies on cerebral ischemia have been performed with young animals, whereas in humans, stroke occurs mainly in the elderly. Consequently, there is little information on whether the cytoskeletal response to ischemia differs between young and old subjects. We found previously that the response of microtubule-associated proteins to stroke is blunted in aged animals [19]. We therefore hypothesized that neurofilament expression would be similarly attenuated in aged animals. To that end, we examined the effects of aging on the response of neurofilaments to focal brain ischemia in rats. 2. Materials and methods Eighteen hours prior to surgery, male Sprague–Dawley rats (n = 12 for 3-month-old rats, weighing 300–360 g, and

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n = 11 for 20-month-old rats, weighing 440–480 g) were deprived of food to minimize variability in ischemic damage that can result from varying plasma glucose levels [11]. Water remained available ad libitum. In all cases, surgery was performed between 8:00 and 13:00 h. The experiments reported in this study were conducted in accordance with the statement regarding the care and use of animals, following the recommendations of Gärtner [9], and were approved by a federal animal care committee. 2.1. Reversible occlusion of the middle cerebral artery Blood flow through the middle cerebral artery (MCA) was temporarily interrupted using a method originally described by Brint et al. [5] and modified by our laboratory [18]. Briefly, animals were anesthetized and a small segment of the skull above the middle cerebral artery was removed 2–3 mm rostral to the juncture of the zygomatic arch and the pars squamosa of the temporal bone. The middle cerebral artery was lifted by a small hook until blood flow through the artery was completely interrupted. Blood flow through the common carotid arteries was then stopped. Three hours later, the middle cerebral artery and the common carotid arteries were reopened, allowing full reperfusion of the brain. After a 7-day survival time, the rats were deeply anesthetized with 2.5% Halothane in 75% nitrous oxide and 25% oxygen, and perfused with buffered saline followed by buffered, freshly depolymerized 4% p-formaldehyde. The brain was removed, post-fixed overnight in phosphatebuffered 4% p-formaldehyde, cryoprotected in 20% sucrose prepared in 10 mmol/l phosphate-buffered saline (PBS), flash-frozen in isopentane and stored at −70 ◦ C until sectioning. 2.2. Histology The brains were cut on a cryostat, and 25 ␮m-thick coronal sections were collected in 4% p-formaldehyde in 100 mmol/l PBS, pH 7.2, post-fixed for 30 min, and stored in polyethylene glycol at −20 ◦ C until use. Every 20th section was histologically differentiated and the area and volume of the ipsilateral cerebral hemisphere and of the infarct site were determined stereologically. The volume of the cortical infarct was expressed as percent of the total volume of the ipsilateral cortex. 2.3. Preparation of cRNA probes The mouse NF cDNA clone for NF-68 was kindly provided by Dr. J.-P. Julien (McGill University, Montreal, Canada) and subcloned into a pBluesscript (pBSSKI(1)+) vector. These plasmids allowed the synthesis of sense and antisense RNA probes. Neurofilament mRNA from mouse

and rat showed homology of about 96%. Northern blot analysis confirmed the specificity for RNA of NF triplet proteins. 2.4. Non-radioactive in situ hybridization A 1.5 kb Digoxigenin-11-UTP-labeled antisense RNA probe was synthesized using a kit supplied by Boehringer Mannheim according to the manufacturer’s specifications. The RNA probe was purified by gel filtration. cRNA quality and quantity were evaluated by detection of the digoxigenin-labeled cRNA with anti-digoxigenin alkaline phophatase, Fab fragment (Boehringer Mannheim) conjugate, followed by color development using the nitro-blue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) system. In situ hybridization was carried out as previously described [18]. Briefly, free-floating sections (60 ␮m) were hybridized in the same buffer containing 50 ng/ml cRNA probe for 6 h at 65 ◦ C. Following high-stringency washes, the slides were incubated in a blocking buffer. Alkaline phosphatase-conjugated sheep anti-digoxigenin-Fab fragments, diluted 1:1000 in blocking buffer, were then applied. For the detection of signal, slides were incubated in a chromogen solution consisting of 330 ␮g/ml NBT, 150 ␮g/ml BCIP and 250 ␮g/ml levamisole in alkaline phosphatase buffer. The reaction was stopped with 10 mmol/l Tris, pH 8.0, plus 1 mmol/l EDTA. 2.4.1. Controls The specificity of the neurofilament antisense RNA probes was assessed by Northern blot analysis. Selected tissue sections also were hybridized with a sense probe. 2.5. Immunocytochemistry Immunocytochemistry was performed as described elsewhere [18]. To block non-specific binding sites, free-floating sections (25 ␮m) were incubated with donkey serum in PBS. Then sections were incubated with mouse monoclonal antibodies recognizing either: [1] NF-68 kDa: clone DA2 1:10; [2] NF-160 kDa: clone NN18 1:400, clone RMO44 1:10, clone RMO281 1:1000; and [3] NF-200 kDa: clone RT97 1:800, clone 3G3 1:800, clone SMI35 1:1000. Afterwards the sections were incubated with biotinylated donkey anti-mouse IgG (Dianova, Hamburg, Germany) diluted 1:200 in PBS. The primary antibody was detected using the ABC system (Vectastain Elite Kit, Vector, Burlingame, CA) and then the antibody complex was visualized with 0.025% 3 ,3 -diaminobenzidine. The primary antibodies were obtained as follows: RT97, NR4, NN18 from Boehringer Mannheim, Germany; RMO281, DA2, RMO44 from Zymed Laboratories Inc., USA; SMI35 from BIOTREND Chemikalien GmbH, Germany and 3G3 from Chemicon Inc., USA. The specificity of the antibodies was verified on Western blots and by

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omission of the primary antibodies in immunocytochemical experiments. 2.6. Binding characteristics of primary antibodies DA2 (anti-NF-68), is a mouse monoclonal antibody that was raised against enzymatically dephosphorylated pig neurofilament protein [23] reacts with the 68 kDa polypeptide of human neurofilaments and specifically recognizes a phosphate-independent epitope [7]. RMO44 (anti-NF-160) binds in a phosphorylation independent manner to the rod domain of NF-M. This antibody also labels neurofibrillary tangles, but only in ETOH/NaCl-fixed tissues [22]. NN18 (anti-NF-160) is a mouse monoclonal antibody directed against the hypophosphorylated human, pig and rat neurofilament [1]. RMO281 (anti-NF-160) binds to a phosphorylated epitope inside the multi-phosphorylation repeat. This antibody labels only processes, not somata [21]. RT97 (anti-NF-200) is a mouse anti-rat monoclonal antibody [26] that binds most strongly to a developmentally-delayed, C-terminal phospho-epitope of bovine NF-H and the equivalent human polypeptide [27] with minor crossreactivity to phosphorylated tau and NF-M [2]. 3G3 (anti-NF-200) has strong reactivity to rat NF-200, and somewhat weaker affinity for NF-200 of other mammalian species. The epitope for 3G3 resides between amino acids 846–1022 of rat NF-H, and the staining is not affected by the level of phosphorylation [3]. SMI35 (anti-NF-200) is reactive with hypophosphorylated neurofilaments [29]. 2.7. Semiquantitative analysis of tissue sections For semiquantitative analysis, coronal sections were collected at three different coronal levels located approximately at −1.2, −3.8 and −4.8 mm from bregma stereotaxic coordinates according to Paxinos and Watson [17]. For one antibody or one RNA probe these sections were in situ hybridized or antibody stained at the same time, respectively. In the infarct border zone the integrated optical densities were measured in a territory with constant size with the KSrun 3.0 software (Zeiss, Germany). Data are presented as percentage of the optical densities on the contralateral side as median ± percentile. Statistical analysis employed the Wilcoxon rank test for comparison of medians between the contralateral and ipsilateral sides, and the Mann–Whitney U-test for age differences, using SPSS software (SPSS Inc., Chicago, IL, USA).

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pronounced inflammation at the infarct site. All of these animals were excluded from further evaluation. Although some blood parameters showed some ageassociated variation, notably blood pressure, the age-related differences were not statistically significant (data not shown). Likewise, there was no significant difference in the volume of cortical infarcts between young and aged rats (40.2 ± 11.4% for the young rats and 40.9 ± 11.3% for the aged rats). 3.1. Neurofilament gene expression Using RNA–RNA in situ hybridization 7 days postischemia, neurofilament-mRNA expression could be demonstrated in the frontal, parietal, perirhinal and piriform cortices of both hemispheres. The staining was concentrated primarily in neurons of cortical layer V (Fig. 1A). There was no gene expression in the necrotic infarct core (Fig. 1C and D). Sections that had been hybridized with a sense-RNA-probe were negative (Fig. 1B). In young animals, some penumbral neurons having clearly increased mRNA expression were evident between cortical layers IV and VI (Fig. 1C). Using semiquantitative analysis (Fig. 2), a 5.1-fold increase (P < 0.05) of neurofilament mRNA expression could be demonstrated in the penumbra, compared to the contralateral side. Some penumbral neurons in older rats also reacted to ischemia with increased NF-mRNA expression (3.1-fold, P < 0.05), but the upregulation was significantly less than in the young animals (P < 0.05). This response in older animals appeared to be limited primarily to neurons of cortical layer V (Fig. 1D). 3.2. NF-68 protein expression 3.2.1. NF-68 expression in healthy cerebral tissue of young and old rats In the present study, only NF-68 (antibody DA2) could be detected simultaneously in somata, axons and dendrites (Fig. 3Db, axon: arrowhead, dendrites: arrows). Age-associated morphological changes were found primarily in the apical dendrites, which were increasingly thickened and bundled in old rats. Some of these processes took on a corkscrew-like shape (Fig. 3Db) or exhibited internal vacuolation with the development of neurofilament-free areas (Fig. 3Da). Here, the normal cytoskeletal architecture seemed disturbed. Furthermore, with increasing age, somatically-localized immunoreactivity shifted from lamina III to lamina V in the frontal cortex (Fig. 3A).

3. Results There was no apparent difference in the clinical status of young and aged rats during the 7-day post-surgical survival time. One 3-month-old rat and two 20-month-old rats died in the first 24 h after MCA occlusion. At the time of perfusion, one young (3 months) and one old (20 months) animal had

3.2.2. Ischemia-related changes in NF-68 expression In contrast to the drastic reduction in protein expression in the infarct core (Fig. 3E), the penumbral neurons in 3-month-old rats exhibited a statistically significant 2.3-fold increase in NF-68 expression compared to the contralateral hemisphere (P < 0.05; Fig. 4A). The immunopositive

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Fig. 1. Neurofilament gene expression 7 days after 3 h occlusion of the middle cerebral artery in young (3 months) and old (20 months) rats. In healthy cerebral tissue, NF-mRNA could be demonstrated in the frontal, parietal, perirhinal and piriform cortices and was localized in perinuclear cytoplasm and in the proximal processes. The neurons of cortical layer V were strongly positive (A). Seven days after cerebral ischemia, gene expression could no longer be detected in the necrotic infarct core (C and D). In contrast, some neurons of the penumbra reacted with clearly upregulated NF-mRNA expression (C and D, arrows). These neurons could be found along the entire infarct margin in young animals, while in old animals they were concentrated in cortical layer V (C compared to D). Control labeling with a sense probe (B), IV, V and VI: standard cortical layer designations. Bars in A–D: 25 ␮m.

Fig. 2. Densitometric analysis of the levels of mRNA expression in the cerebral cortex after cerebral ischemia. Integrated optical densities were measured in the infarct border zone for the two age groups. Data are presented as percent of the optical densities of the contralateral side as median ± percentile (n = 10 for young rats and n = 8 for old rats) (∗ P < 0.05 young vs. old animals).

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Fig. 3. Immunohistochemical detection of NF-68 on thin cryostat sections of young (3 months) and old (20 months) rat brains. The distribution of DA2positive neurons varied in normal frontal cortex of young (Aa) and old rats (Ab). In layers III and V of the frontal cortex of the young animals, NF-68 was localized in the somata (Ca, arrowheads) and dendrites (Ca, arrows). In layer VI, only neuronal processes were detectable (Cb). In the entorhinal cortex, the processes of the perforant pathway were positive (B, arrowheads). Some of the dendrites in old animals showed morphological changes, including neurofilament-free areas in the processes (Db, arrows) and a corkscrew-like structure of the apical dendrites (Db). After MCAO, protein expression in the marginal area of the infarction was increased compared to the contralateral hemisphere (E compared to F), and the positive neurons appeared to have more numerous positive processes (E inset compared to F inset), III, V, VI: cortical layers. Bar in A: 200 ␮m; B, C, E, F: 25 ␮m; D: 10 ␮m.

neurons of the penumbra were found in all cortical layers along the infarction (Fig. 3E). Interestingly, these immunostained neurons exhibited a number of processes (Fig. 3E) and were thereby clearly differentiated morphologically from the neurons of the contralateral, control hemisphere (Fig. 3F). Control neurons exhibited mainly a dipolar morphology and were concentrated in cortical layers III and V (Fig. 3F). Although the ischemia-induced increase in mRNA expression was significantly curtailed in old rats, we did not detect

a statistically significant difference in the immunoreactivity of NF-68 protein between young and old animals. 3.3. NF-160 protein expression 3.3.1. NF-160 expression in healthy brain tissue of young and old rats Antibody RMO44 (Fig. 5A and B) labeled NF-160 independent of its degree of phosphorylation, and allowed the exclusive visualization of neuronal somata (Fig. 5A).

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Fig. 4. Densitometric analysis of the levels of protein expression in the cerebral cortex after cerebral ischemia. Integrated optical densities were measured in the infarct border zone for the two age groups. Data are presented as percent of the optical densities on the contralateral side as median ± percentile (n = 10 for young and n = 8 for older rats) (∗ P < 0.05 and ∗∗ P = 0.01 ipsilateral vs. contralateral side).

Hypophosphorylated NF-160 (antibody NN18; Fig. 5C and D) was localized in axons and dendrites (Fig. 5C). The immunopositive apical dendrites of pyramidal neurons originated mainly from cortical layer III, whence they typically

projected rectilinearly to the upper layers where they ramified heavily (Fig. 5C). The total number of apical dendrites stained with NN18 was lower in old animals, and the dendrites were thickened and bundled in some areas

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Fig. 5. Immunohistochemical detection of NF-160 in ischemic brains of young (3 months) and old (20 months) rats. Protein was identified independent of the degree of phosphorylation (RMO44) in an exclusively somatic localization (A and B). The low-phosphorylated NF-160 (antibody NN18) was axonally and dendritically (C and D arrowheads) localized, and the highly phosphorylated NF-160 (RMO281; E and F) was exclusively axonal. The age-associated changes in the morphology of the NN18-positive apical dendrites were remarkable (Da compared to C). In aged rats, the dendrites frequently had a bundled appearance (Da, arrowheads) and exhibited internal vacuolation (Da, inset). Comparable changes were also seen in apical dendrites in the marginal area of the infarction 7 days after MCAO (Db), including NF-160-free areas in the processes and a corkscrew-like conformation of apical dendrites (Db). The RMO281-positive axons were particularly abundant in the penumbra and formed a dense tangle of processes (Fa and b); II, III: cortical layers. Bars in A–F: 25 ␮m; insets, 10 ␮m.

(Fig. 5Da); furthermore, more abundant vesicular, neurofilament-free areas were visible in apical dendrites of aged rats (Fig. 5Da). For hyperphosphorylated NF-160 (antibody RMO281; Fig. 5E and F), immunoreactivity was associated with axons of the perforant pathway (Fig. 5Eb), whereas dendrites were not visualized, leading to the conclusion that NF-160 is localized mainly in axons.

3.3.2. Ischemia-related changes in NF-160 expression Semiquantitative analysis (Fig. 4B) of RMO44-staining showed a 40% reduction of immunoreactivity in the penumbra of young animals (P < 0.05) compared with the contralateral, non-infarcted side (Fig. 5B compared to A). In old animals, NF-160 expression in the marginal zone decreased by 50% (P < 0.05). This result was surprising, because a statistically significant reduction of neurofilament

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expression in the penumbra was not seen with any other antibody. At the same time, RM044 was the only antibody that exclusively detected somatically-localized neurofilaments. The intensity of the NF-160 protein expression did not vary significantly between the age groups. For the low-phosphorylated NF-160 (antibody NN18), clearly increased immunoreactivity (2.5-fold increase; P = 0.01; Fig. 6) was detected 7 days after MCAO in the

penumbra of the 3-month-old rats compared to the contralateral control hemisphere. This increase could be attributed to increased immunoreactivity in the apical dendrites of the pyramidal neurons (Fig. 5Db compared to C). Some apical dendrites were thickened, bundled and vacuolated. Thus, they exhibited the same morphological changes as the NN18-positive apical dendrites of aged animals without ischemic damage (Fig. 5Da compared to b).

Fig. 6. Immunohistochemical detection of NF-200 on thin cryostat sections of ischemic brains of young (3 months) and old (20 months) rats 7 days after ischemia. The distribution of NF-200 varied as a function of the phosphorylation degree of the protein after labeling with different antibodies. Antibody 3G3 recognized a phosphorylation-independent epitope (A and B). The protein was localized in dendrites (Aa and b arrows). With increasing age, more neuronal somata became visible (Ab, arrowheads) and the staining of fine, tangentially-oriented processes (Aa, arrowheads) decreased. Antibody SMI35 recognized hypophosphorylated NF-200 (C and D), and RT97 recognized the hyperphosphorylated protein (E and F). Both proteins were axonally localized. There were very fine, SMI35-positive processes in cortex lamina III (Ca), which were not stained with RT97. (B, D, and F) The changes in protein expression after MCAO in the penumbra (Bb, Db, Fb) compared to the contralateral hemisphere (Ba, Da, Fa). II, III, V: cortical layers. Bars in A–F: 25 ␮m.

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The highly phosphorylated NF-160 (antibody RMO281) was slightly, and non-significantly, upregulated in neuronal processes of penumbral neurons in both age groups 7 days after MCAO (Fig. 4B). In the marginal infarction zone, tangentially oriented fibers with an altered morphology were more frequent, especially in young animals (Fig. 5Fb compared to a). These fibers formed a dense tangle, with some processes appearing thickened. No age-associated differences could be detected. 3.4. NF-200 protein expression 3.4.1. NF-200 expression in healthy cerebral tissue of young and old rats The antibody clone 3G3 labeled NF-200 independent of the degree of protein phosphorylation (Fig. 6A and B) and stained dendrites and neuronal somata. The apical dendrites (Fig. 6A, arrows) of pyramidal neurons of cortical layer III extended all the way to cortical layer II in the frontal cortex, where they ramified extensively. In addition, very fine, rectilinear and tangentially-oriented processes were revealed in laminae II through V (Fig. 6Aa, arrowheads). With increasing age, 3G3-positive neuronal somata were more frequent (Fig. 6Ab compared to a). The perforant pathway, as an example of an axon-rich pathway, could not be visualized with this antibody. Hypophosphorylated NF-200 (antibody SMI35, Fig. 6C and D) was found only in neuronal processes. These had an open structure in cortical layer II (Fig. 6Ca) and, judging from their shape and orientation, were not apical dendrites. In the entorhinal cortex, SMI35-immunoreactivity was associated with axons of the perforant pathway (Fig. 6Cb). Antibody clone RT97 recognized highly phosphorylated NF-200 and also exclusively labeled neuronal processes, but no apical dendrites could be seen (Fig. 6E). Linearlyextending processes were only rarely visualized. Instead, especially in laminae V and VI, fine, intensely-stained processes without a recognizable primary orientation appeared, but only short segments of these were visible, resulting in a punctate staining pattern (Fig. 6Eb). 3.4.2. Ischemia-related changes in NF-200 expression The antibody against a phosphorylation-independent epitope of NF-200 (3G3) revealed a significant increase in immunoreactivity in the marginal zone of young as well as old animals. This increase could be attributed to intensified staining of the apical dendrites of penumbral neurons (Fig. 6Bb). In addition, however, more numerous tangentially oriented fibers occurred in the penumbra (Fig. 6Bb, arrowheads). The intensified immunoreactivity in the marginal infarction zone was slightly greater in old animals than in young ones (1.9-fold compared to 1.4-fold; Fig. 4C), but the difference between the age groups was not statistically significant. One week after cerebral ischemia, the expression of hypophosphorylated neurofilament 200 (antibody SMI35) in

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the ipsilateral penumbra of young and old animals was 2X greater than in the contralateral control hemisphere (Fig. 4C). In young animals, this heightened immunoreactivity was statistically non-significant due to high variability. This increased expression was accompanied by a proliferation of axonal processes, similar to that seen for hyperphosphorylated NF-160 (Fig. 6Db compared to a). The greatest upregulation of protein expression after cerebral ischemia was measured for the highly phosphorylated NF-200 (antibody RT97). Young animals reacted to the infarction with a 5.1-fold augmentation of expression, and older animals showed a 6.2-fold increase (P < 0.05; Fig. 4C). Here, too, the rise manifested itself as both an increase in the immunopositive processes and in their more intense staining (Fig. 6Fb compared to a).

4. Discussion The results of this study show that NF-mRNA synthesis is significantly increased in the penumbra of old and young rats following temporary, 3 h occlusion of the middle cerebral artery. The infarct-induced upregulation of NF-mRNA was significantly greater in young animals than in old animals. Our data thus indicate that neurofilament protein biosynthesis is augmented in reaction to the inflicted trauma, similar to the upregulation of NF-mRNA seen in the peripheral nervous system following axotomy [25]. We used immunocytochemistry to enable the precise anatomical localization of changes in protein expression in the infarcted hemisphere. Despite a significant influence of age on NF-mRNA upregulation, we did not detect a corresponding age-effect on neurofilament protein levels immunocytochemically. A Western blot analysis or enzyme-linked immunosorbent assay (ELISA) might provide a more sensitive measure of quantitative protein changes. These methods, however, are problematic because changes in the penumbra would be diluted or obscured by the inclusion of surrounding tissue, including the infarct core. An elegant solution to this problem in future studies might be the use of microdissection to excise the areas of interest from a single cryostat section for quantitative analysis [4]. Microscopic analysis of the infarct penumbra reveals that the increase in neurofilament immunoreactivity correlates with two distinct morphological changes. For the axonally-localized proteins (hyperphosphorylated NF-160, high- and low-phosphorylated NF-200) in the penumbra, there was a clear increase in the numerical density of thin processes, and in the immunostaining intensity of individual fibers. This finding, in the context of the involvement of neurofilaments in neuroplastic processes [13,14,16,20,28] could be interpreted as the localization of these neurofilament proteins in newly-sprouting axon collaterals following damage to the neurons [6]. With regard to this subject there are indications in the literature that a trauma can entail neuronal destruction leading to loss of axonal connections

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and synapse destruction. The surviving deafferentiated neurons react to this event by sprouting of axonal collaterales [6]. These are subsequently elongated, the contact with another nerve cell results in synaptogenesis, and the resulting connection is firmly established [12]. The neurofilament proteins are involved in all of these plasticity processes. It could be shown that NF-68 is expressed in axonal growth cones [13]. NF-M and NF-H are involved in the stabilization of the ramification of newly sprouted axons and their elongation [20,28], while NF-H is said to play an important role primarily in the stabilization and maturation of already existing connections [16,14]. On the other hand in the infarct penumbra, we found an increase in dendritically-localized protein immunoreactivity (NF-68, hypophosphorylated NF-160) in apical dendrites that was always coupled with morphological changes in the dendrites such as thickening and vacuolation. Similar dendritic changes were also found in the contralateral cortex of aged animals and appear to be primarily age-associated, degenerative changes. This interpretation is consistent with reports that, at an advanced age, trauma or damage by free oxygen radicals can lead to a degradation of the neurofilament network, disrupting axonal and dendritic transport and resulting in the accumulation of neurofilaments [10,13]. Based on these results, it becomes apparent that the participation of neurofilament proteins in post-ischemic processes in the penumbra is quite complex. Neurofilament proteins not only accumulate in injured, degenerating neurons, but they are involved in the reorganization of connectivity by surviving penumbral neurons as well. In a previous study [19] we have shown similar age-dependent results for the microtubuli-associated proteins 2 and 5 (MAP2 and 5). Focal cerebral ischemia resulted 1 week after the stroke in the border zone adjacent to the infarct of 3- and 20-month-old male Sprague–Dawley rats in a vigorous expression of MAP1B and its messenger ribonucleic acid, as well as MAP2 protein, respectively. Although the morphologic features of fibers in the infarct border zone were similar in both age groups, the upregulation of these key cytologic elements was generally diminished in aged rats. These results suggest that the regenerative potential of the aged rat brain appears to be competent, although attenuated. Our study has shown that neurofilament gene expression was significantly different between young and aged animals. Furthermore, we have shown that neurofilament proteins are involved in the response of the brain to ischemia. Using mentioned methods, we found no significant differences in protein expression between the two age groups. Further studies with sophisticated methods, e.g. microdissection will be necessary to understand how changes in production, phosphorylation and distribution of specific neurofilament proteins influence the plasticity of brain tissue in response to acute injuries such as head trauma and stroke.

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