Ependyma as a Possible Morphological Basis of Ischemic Preconditioning Tolerance in Rat Spinal Cord...

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Cellular and Molecular Neurobiology [cemn] pp1172-cemn-484794 March 25, 2004 21:20 Style file version Oct 23, 2000

Cellular and Molecular Neurobiology, Vol. 24, No. 3, June 2004 ( C© 2004)

Ependyma as a Possible Morphological Basis of IschemicPreconditioning Tolerance in Rat Spinal Cord IschemiaModel: Nestin and Fluoro-Jade B Observations

Judita Orendacova,1,3 Eniko Racekova,1 Karolına Kucharova,1

Barbora Pousova,1 Tomas Ondrejcak,1 Marcela Martoncıkova,1

Zuzana Daxnerova,2 and Jozef Marsala1

Received September 30, 2003; accepted November 13, 2003

SUMMARY

1. To test our hypothesis that a transient nonlethal ischemic insult benefits the lum-bosacral spinal cord ischemic injury, nestin, the marker of proliferating cells, and Fluoro-JadeB, the marker of degenerating cells, were used in rats. Morphological outcome was evalu-ated after 12-min ischemia versus 12-min ischemia preconditioned by 3-min ischemic periodand 30-min recirculation (IPC), in each group followed by 2, 3, and 4 days of posttreatmentsurvival.

2. Twelve-minute ischemia, inducing nestin-positivity in ependyma and reactive astro-cytes at the L1−3 spinal cord segments, shows this region as the viable region of spinal cord inall postischemic survival periods. On the other hand, abundance of Fluoro-Jade B-positivecells, distributed throughout the dorsal horn and intermediate zone of L4–S2 segments,points out the most injured spinal cord region by ischemia.

3. After the same ischemic insult in IPC rats only a few nestin-positive ependymalcell and reactive astrocytes appeared beside the nestin-positive vessels in the lower lumbarand sacral spinal cord segments of all survival periods. The appearance of nestin-positivecells in the spinal cord segments, which “should have been affected” by ischemia indicatesprotection of this region by the IPC treatment.

4. The number and density evaluation of Fluoro-Jade B fluorescent cells of L4–S2 seg-ments after ischemia and IPC confirmed that degenerating cells were significantly reducedin the IPC rats in all survival periods.

5. Our results showing the immunohistochemical response of epemdyma, committedto the presence of viable tissue, indicate that the ependymal cells may contribute to theischemic resistance in the IPC rats.

KEY WORDS: ischemia; ischemic preconditioning; ependyma; nestin; Fluoro-Jade B;quantitative evaluation.

1 Institute of Neurobiology, Slovak Academy of Sciences, Kosice, Slovak Republic.2 Institute of Biology and Ecology, Faculty of Sciences, P.J. Safarik University, Kosice, Slovak Republic.3 To whom correspondence should be addressed at Institute of Neurobiology, Slovak Academy of Sciences,

Soltesovej 4, Kosice 040 01, Slovak Republic; e-mail: [email protected].

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0272-4340/04/0600-0477/0 C© 2004 Plenum Publishing Corporation

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478 Orendacova et al.

INTRODUCTION

In lower vertebrates, the spinal cord ependyma plays a significant role in neuronalregeneration. In these animals the ependymal cells rapidly proliferate, migrate, anddifferentiate to regenerate the injured spinal cord. They extend long processes thatare believed to provide longitudinal channels to guide regenerating central axonscaudally (Simpson and Duffy, 1994). Ependymal proliferation in response to spinalcord injury has been also described in morphological studies using adult mammals(Beattie et al., 1997; Vaquero et al., 1981; Wallace et al., 1987). However, it is notknown if these proliferating ependymal cells have similar beneficial regenerativeeffect as those in the lower vertebrates. In the adult rat brain, nestin, a neuralstem cell protein, can be found only in endothelial and selected subventricular cells(Morshead et al., 1994; Tohyama et al., 1992). Some evidence suggests that stem-likecells can be localized in or near the ependymal layer of the brain and spinal cord(Chiasson et al., 1999; Doetsch et al., 1999; Garcia-Verdugo et al., 1998; Horner et al.,2000; Johansson et al., 1999). Although Weiss et al. (1996) demonstrated multipo-tent stem cells, which can be isolated, expanded, and differentiated in vitro in theadult mice lumbosacral spinal cord (Shihabuddin et al., 1997) the normal ependymashows no immunoreactivity to nestin, a marker for neural precursor cells (Mokry,1999).

On the basis of experimental interventions mimicking spinal cord injuries, thefindings on nestin-positivity were found to be different from those mentioned above.Frisen et al. (1995) proposed an interesting hypothesis that a precursor cell popula-tion, which can be identified by nestin expression, exists around the central canal,and can proliferate and migrate to the injury site and contribute to the glial scar.The spinal cord, injured by clip compression, demonstrated a dramatic increase inependymal proliferation 3 days after injury and concomitant strong nestin expres-sion in ependyma (Namiki and Tator, 1999). Nestin expression was present in someneurons localized in the outer boundary zone of the ischemic lesion in the cortexand striatum, and in most ependymal cells in the ventricular and subventricular zonefrom the second day after middle cerebral artery occlusion (Li and Chopp, 1999).After a stroke, nestin immunoreactivity in glial, neuronal, and ependymal cells issuggestive of protein expression pattern found in the developing brain.

Ischemic preconditioning (IPC) has been defined as an endogenous cellularprotective mechanism, evoked by a brief ischemic period. IPC renders the CNStissue more resistant to the subsequent lethal ischemic insult (Burda et al., 2003;Danielisova et al., 2003; Kitagawa et al., 1991; Lukacova, 1999; Nemethova et al.,2003; Orendacova et al., 2003; Radonak et al., 2003; Zvara et al., 1999). A similarprotective effect of IPC has been observed after experimental traumatic brain injury(Perez-Pinzon et al., 1999). Contrary to the cerebral trauma, IPC does not improvethe outcome after spinal cord contusion injury, and even worsens the damage to thespinal cord white matter in rat (Ondrejcak et al., 2003).

A novel fluorochrome Fluoro-Jade B, like its predecessor Fluoro-Jade, is ananionic fluorescein derivative useful for histological staining of neurons undergoingdegeneration (Schmued et al., 1997; Schmued and Hopkins, 2000). It is a reliabletracer of high affinity, which makes it easier to detect labeled fine neuronal processes

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Ischemic Preconditioning Benefits Ischemic Injury 479

including dendrites and axons. To date, we know of no published studies using thisfluorochrome for detection of degenerating cells after ischemic injury.

In the present work we used nestin and Fluoro-Jade B labeling to study theregional differences between ischemic and IPC-induced changes of ependyma andthe gray matter of rat lumbosacral spinal cord.

MATERIALS AND METHODS

The experimental procedure was performed with approval from the AnimalCare Committee of the Institute of Neurobiology.

Surgical Procedure

Wistar albino rats were anesthetized with 1.5% halothane in a 45% oxygenair mixture. The tail artery was cannulated with a PE-50 polyethylene catheter formonitoring the mean distal aortic blood pressure (MDAP). The left femoral arterywas isolated and a Fogarty 2F balloon-tipped catheter was introduced for later ad-vancement into the descending thoracic aorta. The left internal carotid artery wascannulated with a 20-gauge catheter for measurement of the mean proximal aorticblood pressure (MPAP); this cannula was connected to a head blood-collection cir-cuit (37.5◦C) that included an 88-cm vertical column of heparinized (1 U/ml) salinesolution. When the aorta was occluded, proximal aortic blood was allowed to bleedinto the heparinized column, maintaining MPAP at 65 ± 3 mmHg. To induce spinalcord ischemia, the Fogarty 2F catheter (situated in the left femoral artery) was in-serted retrogradely into the descending thoracic aorta 10.5 cm from the femoralarteriotomy. During the occlusion the catheter was inflated with 0.05 ml of salinesolution, and aortic occlusion was confirmed by reduction in MDAP. (1) Rats of theischemic group were subjected to the 12-min occlusion of thoracic aorta. (2) Rats ofthe ischemic preconditioned group (IPC) were first subjected to the 3-min occlusionfollowed by 30-min reperfusion, and then again to the 12-min occlusion of thoracicaorta. Then, the vented blood from the carotid artery cannula and the heat exchangerwas returned, the catheters removed, the surgical wounds closed, and the rats of bothexperimental groups put to recovery.

Tissue Sampling for Immunohistochemical and Fluorescent Analysis

The animals were subjected to neurological testing (ambulation and stepping)after two, 24 hr and at the end of each survival period, i.e. 2, 3, and 4 days, for eval-uation of the hind limb motor function. The animals without loss of motor functionafter ischemia or with loss of motor function after IPC were discarded. The rats wereperfused with 4% paraformaldehyde in a deep anesthesia. The lumbosacral spinalcord was removed from the backbone, postfixed, cryoprotected in 30% sucrose andfrozen 40µm thick coronal sections were cut and collected in phosphate buffer (PB).

After blocking in normal serum, the free-floating sections were treated withthe primary monoclonal antibody (mAb) anti-nestin Rat-401 (Developmental

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Hybridoma Studies Bank) diluted 1:400. After thorough washing, the sections wereincubated with antimouse IgG (Dako) secondary antibody. Diaminobenzidine(DAB) was then used as a sensitive chromogen for light microscopic visualization ofnestin-positive cells.

Fluoro-Jade B (Histo-Chem Inc.) was used as a marker of neurons undergoingdegeneration. The sections mounted on 2% gelatin coated slides and air-dried wereheated at 50◦C for at least half an hour before staining. The slides were immersed inabsolute alcohol for 3 min, for 1 min in 70% alcohol, and for 1 min in distilled water.Then the slides were transferred to the solution of 0.06% potassium permanganatefor 15 min and rinsed in distilled water for 2 min. After 30 min in the staining solution,prepared according to the Schmued and Hopkins (2000), 3× 1 min rinses in distilledwater followed. The slides were dried at room temperature, cleared by xylene, andcover slipped with Fluoromont. The sections were observed by Olympus BX51 lightmicroscope.

Some alternate sections were processed according to the Gallyas reduced silvermethod for degenerating neurons (Gallyas et al., 1980).

Quantitative Assessment

The sections were transformed into digital images by the Olympus ReflectedFluorescence system U-RFL-T, the Olympus BX51, the digital camera DP50, andthe visual communication suite Olympus DP-soft, version 3.0. To determine thenumber and optical density of Fluoro-Jade B-exhibiting neurons in the L4–S2 spinalcord the UTHSCSA Image Tool and Microsoft Excel were used in the rats surviving4 days after ischemia (n = 3) and after IPC (n = 3). The gray scale values relativeto the highest concentration of Fluoro-Jade B in degenerating neurons and the lackof the fluorescent marker in the section was defined as the 100 and 0% density values.The results were statistically evaluated by Dunn’s Method and Tukey Test and havebeen given as means ± SD.

RESULTS

Ischemia

In the most injured L4–S2 spinal cord segments ischemia caused widespreadneuronal necrosis, which appeared as distinct demarcated area throughout the dorsalhorn and intermediate zone on Gallyas impregnated sections (Fig. 1(A)). Pathomor-phological signs of degeneration detected along the rostro-caudal extent of the mostinjured segments increased from the second postischemic day and were fully devel-oped after 4-day survival. Nestin immunohistochemistry did not show characteristicexpression pattern either of vessel walls or of ependymal or glial cells (results notshown).

The L3 spinal cord segments, comprising the immediate rostral border of thelesioned region, revealed distinct nestin immunoreactvity of the ependymal cellsand reactive astrocytes in all postinjury survival periods. The ependymal wall was

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Fig. 1. Representative micrographs of Gallyas silver impregnation in L4–S2 segments after 4-daysurvival. (A) Pale circumscribed area of degenerated cells in the dorsal horn (DH) and the inter-mediate zone (IZ) after 12-min ischemia. (B) The same region without any signs of degeneration,either in the dorsal horn (DH) or in the intermediate zone (IZ), after IPC. Central canal (CC), bar =400 µm.

visible as a dark layer of varying thickness around the spinal cord central canal. Theapical cytoplasm of the ependymal cell was reduced and the nuclei appeared to belocated closer to the lumen of the central canal (Fig. 2(A)). The ependymal cells werecharacteristic by their long nestin-positive processes extending radially from the cellbasal pole and traceable for a long distance from the cell bodies. At the luminal sur-face of ependyma short processes progressed to the opposite side forming bridgednetwork in the central canal lumen (Fig. 2(B)). The reactive astrocytes, character-ized by their starry shape body due to multiple running processes, expressed nestin(Fig. 2(C)). The vessels in the immediate border to the necrotic zone were alsonestin-positive. In general, the immunohistochemical observation of lumbosacralspinal cord in rats exposed to 12-min ischemia showed that injured spinal cordsegments were rostrally delineated by a region of characteristic nestin immunore-activity.

Fluoro-Jade B fluorescence exhibited a different distribution pattern in compar-ison to the distribution pattern of nestin-positivity mentioned above. DegeneratedFluoro-Jade B-positive cells were observed at L4–S2 spinal cord segments, i.e. in seg-ments injured by ischemia and were characterized by a conspicuous green fluorescentlabeling distributed throughout the dorsal horns and intermediate zone except mo-toneurons in the ventral horns (Fig. 4(A)).

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Fig. 2. Twelve-minute ischemia: Representative micro-graphs of nestin-immunoreactivity in the L3 segment (ad-jacent to the lesioned segments) after 4-day survival. (A)Flattened ependymal wall visible as a dark layer aroundthe central canal (CC). Nestin-positive ependymal cellscharacterized by their long radially running processesinto the gray matter. Bar= 100 µm. (B) Short processesat the luminal surface form bridged network in the cen-tral canal (CC). Bar = 50 µm. (C) Abundant of reactiveastrocytes (arrows) in the gray matter. Bar = 100 µm.

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Ischemic Preconditioning

In the L4–S2 spinal cord segments there were no signs of degeneration afterGallyas silver impregnation (Fig. 1(B)). Nestin-immunohistochemistry showed char-acteristic labeling of vessels. Nestin-expressing ependymal cells appeared randomlyin L4–S2 segments in all survival periods. The ependymal wall clearly displayed its nor-mal multilayered morphology, and no irregularities in thickness could be detected asdescribed after ischemia (Fig. 3(A)). Nuclei of ependymal cells were located basallyand the apical cytoplasm surrounded continually the lumen of the central canal.The labeled radially running processes of ependymal cells were fewer and shorter incomparison with that visible after 12-min ischemia (Fig. 3(B)). Nestin-positivity ofreactive astrocytes occurred very rarely. Nestin-positive vessels were seen throughtthe lumbosacral spinal cord segments in all survival periods (Fig. 3(C)).

Fluoro-Jade B fluorescence showed only mild presence of the degenerating cellssituated preferentially in the intermediate zone. The degenerating cells were rarewithin the L4–S2 spinal cord segments (Fig. 4(B)). The Flouro-Jade B findings ofdegenerated cells are in accordance with demonstration of postischemic spinal corddegeneration by the silver impregnation (Fig. 1(B)).

Quantitative Assesment

Quantitative analysis of Fluoro-Jade B sections showed a significant increaseof fluorescent cells throughout the gray matter of L4–S2 spinal cord segments afterischemia in comparison to the IPC rats (Table I). In the rats submitted to the 12-min ischemia the highest number of Fluoro-Jade B-exhibiting cells were present inthe dorsal horn and intermediate zone. In the rats preconditioned by ischemia onlynegligible number of Fluoro-Jade B-expressing cells appeared and they were mostlydistributed in the intermediate zone. Distribution of Fluoro-Jade B fluorescent cellsin the upper lumbar spinal cord segments was of no importance, therefore it wasnot evaluated quantitatively. The optical density values, reflecting concentration ofFluoro-Jade B in degenerating cells, were 63.15 ± 1.17. These values were the samein the rats after ischemia and IPC. There were no significant differences between 2-,3-, and 4-day survival.

DISCUSSION

Ischemia and Ischemic Preconditioning

Although the recent research in the ischemic myocardium, brain, and spinal corddemonstrates different degree of the tissue protection with IPC involving longerreperfusion intervals ranging from 1 to 5 days (for review, see Lukacova, 1999),shorter reperfusion intervals ranging from 10 min to 6 hr also offers some degreeof neuroprotection, thus suggesting that several mechanisms may play a role in IPCprotection of the nervous tissue. The present work investigated whether 30-min reper-fusion after 3-min ischemia is effective against ischemic injury. To address this ques-tion, we used a reliable rat model of spinal cord ischemia (Taira and Marsala, 1996).

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Fig. 3. Ischemic preconditioning: Representative micro-graphs of nestin-immunoreactivity in L4–S2 segments after4-day survival. (A) Ependymal wall shows its normal mul-tiplayer morphology. (B) Ependymal cells radiate few shortprocesses into the gray matter. (C) Nestin-positive vesselscharacterize the control adult nervous tissue. Central canal(CC), bar = 100 µm.

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Fig. 4. Representative micrographs of Fluoro-Jade B-labeling in the L4–S2 segments after 4-day sur-vival. (A) Twelve-minute ischemia induces fluorescence throughout the dorsal horn and intermediatezone. (B) IPC-induced scant fluorescence in the intermediate zone. Central canal (CC), bar = 200 µm.

With this model it was showed that ischemic intervals longer than 8-min trigger arapid degeneration of spinal cord interneurons and detectable neurohistopathologi-cal (Marsala et al., 1997) and histochemical (Yang et al., 2000) changes in lumbosacralspinal cord segments. It was shown that 12-min ischemia is sufficient to induce spasticparaplegia in all experimental animals and 2-day survival postoperation was crucialfor development of either the neurological injury in ischemic rats (Ondrejcak, 2001),or the immunohistochemically detectable protective effect of IPC. Silver impreg-nation analysis of neuronal damage and concurrent histochemical characterizationof NADPH-diaphorase positive neuronal pools in the rabbit lumbosacral segmentsshowed extensive neuronal degeneration in the central gray matter between 1 and4 days after ischemia (Marsala et al., 1997).

Distribution of Nestin-Positive Cells

Hockfield and McKay (1985), who first produced the Rat-401 anti-nestin mon-oclonal antibody, identified cell types immunoreactive for nestin. Nestin, an inter-mediate filament protein, found in the neuroepithelial precursor cells can also beexpressed in vitro in the multipotent precursor cells in the adult CNS (Gritti et al.,1996; Reynolds and Weiss, 1992; Weiss et al., 1996). Although the normal ependyma

Table I. Number of Fluoro-Jade B-Exhibiting Neurons FollowingIschemia (I) and Ischemia With Ischemic Preconditioning (IPC)Distributed Throughout the Gray Matter of L4 Segment after 4-Day

Survival

Dorsal horn Zona intermedia Ventral horn

I 160.13 ± 5.73 123.07 ± 3.37 76.14 ± 8.76IPC 7.79 ± 2.09∗ 17.48 ± 8.81∗ 5.33 ± 1.52∗

Note. Values are expressed as means ± SD in the section of 10 rats.The results were evaluated by Dunn’s Method and Tukey Test.∗P < 0.001.

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of the central canal maintained very limited cell proliferation (1.5% of the cells areproliferating), it did not exhibit nestin immunoreactivity, which would indicate pre-cursor cell qualities (Namiki and Tator, 1999). Since it has been reported that allof the known multipotent neural stem cells express nestin (Dahlstrand et al., 1995;Stemple and Mahanthappa, 1997), we used nestin immunoreactivity to identify cellswith precursor cell qualities in the spinal cord ependyma of adult rats after ischemicinjury.

Expression of the stem cell marker, nestin, by the ependymal cells and by reactiveastrocytes indicates that these differentiated cells have potential of dedifferentiation.This attribute was considered to be characteristic for viable tissue distributed mainlyin the vicinity of injury. Therefore, our findings that after ischemic injury, nestin-positivity was found only in the areas adjacent to the lesion characterize this regionas viable noninjured. Similarly, the occurrence of nestin-positivity in the ependymalcells and reactive astrocytes after IPC in those spinal cord segments (L4–S2), which“should have been affected” by ischemia, confirms the viability of this region, i.e.indicate protective effect of short ischemic preconditioning. Our data, showing theperilesion distribution of nestin expression after ischemia, are consistent with similarobservations after a clip-compression spinal cord injury (Liu et al., 2002; Namiki andTator, 1999). Even though the mechanism of nestin reexpression is unclear, the classicstatic view of the glial scar is challenged by the observation of reactive astrocytesexpressing nestin. Kaya et al. (1999) consider the possibility that nestin expressionmay contribute to the dynamic feature of glial scar formation, and Duggal et al.(1997) have suggested that nestin may also facilitate the formation of new synapticconnections in neurons in the immediate neighborhood of the glial scar.

Distibution of Fluoro-Jade B Degeneration

Fluoro-Jade B, a novel fluorochrome, is demonstrated to be high-affinity trac-ers detecting neuronal degeneration (Schmued et al., 1997; Schmued and Hopkins,2000). It allows a rapid, reliable, and sensitive labeling of fine neuronal processesincluding distal dendrites, axons, and terminals resulting in a yellow-green appearingfluorescence in ultraviolet excitation. Although kainic acid exposure was the onlyneurotoxic insult systematically examined by this fluorochrome, it is believed thatthis fluorescent tracer will detect dead neurons regardless of the cause of death. Pu-tative chemistry and mode of Fluoro-Jade B action remains to be confirmed, butregardless of the mechanism of action, the authors discuss that the tracer can be usedto resolve a number of questions involving neuronal cell death, including studies onbrain pathology, toxicology, development, dementia, and neuroprotection.

Recently we have successfully used Fluoro-Jade B to label the dying cells withinrostral migratory stream (RMS) at various postnatal periods (Martoncıkova et al.,2003) as well as in adult rats after olfactory bulbectomy, i.e. in the absence of themigrating cells target (Pousova et al., 2003). Use of Fluoro-Jade B after 12-min is-chemia resulted in labeling numerous degenerating cells throughout the gray matterof lumbosacral spinal cord segments. The presented distribution of ischemia-induceddegeneration correlates with the ischemic neuronal damage detected by some re-cently developed silver impregnation procedures after Gallyas (Gallyas et al., 1980)

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and Nauta (Marsala et al., 1989). Although the qualitative observation of the Fluoro-Jade B-labeling was plausible, we evaluated the nuber and density of degeneratedcells by the methods used in some previous papers (Kucharova et al., 2001; Marsalaet al., 2003; Maslej et al., 2003). This approaching put an evidence of significant de-crease of degenerating cells number after IPC. The published quantitative assessmentwas provided in animals of both experimental groups after 4-day survival, becausethe differences between 2, 3, and 4 days were negligible.

CONCLUSIONS

Nestin-immunohistochemistry indicates significance of the spinal cord region inthe vicinity of damaged tissue.

Fluoro-Jade B appears to be a reliable neuropathological technique comparablewith the silver impregnation allowing demonstration of the IPC benefit effect.

ACKNOWLEDGMENTS

This work was supported by the VEGA grants: 2/2082/22, 2/3216/23, 2/3217/23,and 2/3218/23; the APVT grant: 51-013002. The mAb Rat-401, developed by S.Hockfield, was obtained from the Developmental Studies Hybridoma Bank NICHD,Department of Biological Sciences, University of Iowa, Iowa City, IA 52242.

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Ependyma as a Possible Morphological Basis of Ischemic Preconditioning Tolerance in Rat Spinal Cord...

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