Proliferation and Regeneration of Retrogradely Labeled Adult Rat Corticospinal Neurons in Culture

Experimental Neurology 170, 277–282 (2001) doi:10.1006/exnr.2001.7705, available online at http://www.idealibrary.com on

Proliferation and Regeneration of Retrogradely Labeled Adult Rat Corticospinal Neurons in Culture Arshak R. Alexanian and Howard O. Nornes Department of Anatomy and Neurobiology, Colorado State University, Fort Collins, Colorado 80523 Received November 27, 2000; accepted March 30, 2001

These are the first studies to demonstrate that adult rat corticospinal tract (CST) neurons, which were identified by retrograde neuronal labeling, retain regenerative and proliferative potential. To determine if adult CST neurons undergo cell division, we tested if these retrogradely labeled cells synthesize DNA by adding BrdU to the cultures 24 h prior to fixation of the cells. The result shows that adult corticospinal tract neurons are capable of DNA synthesis, and our total cell counts with labeled cells counts further suggest that these cells undergo cell division. ©

rounds of mitotic proliferation. The interpretation of this work, however, needs further clarification because it is unclear whether these dividing cells are derived from adult neurons that reenter the mitotic cycle, from stem cells which differentiated, or from both simultaneously. Here we report data which demonstrate that adult corticospinal tract neurons are capable survival, proliferation, and neurite outgrowth. MATERIALS AND METHODS

Retrograde Labeling of Corticospinal Neurons

2001 Academic Press

Key Words: adult neurons; retrograde labeling; proliferation; regeneration; BrdU.

INTRODUCTION

Until recently, scientists assumed that shortly after birth, neurons loose their ability to grow and cells that die cannot be replaced. However, in the past few years, it has been shown that certain kinds of cells can grow in adult brains, including those of humans (5, 8, 10). It has become clear that the central nervous system (CNS) has the capacity to regenerate all neural cell types: neurons, astrocytes and oligodendrocytes (8, 9). Recently, one of the main research strategies for regeneration of CNS has focused on stem cells, which are the natural units of embryonic generation. These cells also have a major role in regeneration processes of a variety of tissues of adults, including both central and peripheral nervous system (4, 6). In the adult CNS of mammals, a dividing population of stem cells in some regions of brain is capable of limited cell replacement that can occur both under natural and traumatic conditions (7, 10). Thus, the role of cellular division as it applies to the adult nervous system has centered primarily on the proliferative powers of stem cells. Recently, Brewer (3) presented evidence that adult neurons are also capable of survival and proliferation in culture. He cultured adult rat hippocampal cells and showed that cells with morphological and immunocytochemical characteristics of neurons undergo multiple

The experiments were performed on adult male Sprague–Dawley rats. Rats (190 –220 g) were anesthetized with isoflurane. The skin and underlying muscles were cut and a laminectomy was performed at the level C1–C2 of the cervical cord. A volume of TMR-DA (tetramethylrhodamine dextranamine of MW 10,000 or 3,000; Molecular Probes, D1817 or D-3308, respectively), as well as of other tracers such as Oregon Green (Molecular Probes, D-7171, 10,000 MW), dextran fluorescein and biotin (Molecular Probes, D7178, 10,000 MW), dextran fluorescein (Molecular Probes, D1820, 10,000 MW), and Fast Blue (Sigma, F5756) were injected bilaterally into the spinal cord (0.5 ␮l per injection), just lateral to the midline by use of Hamilton syringe. All tracers were dissolved in PBS (pH 7.0) except TMR-DA. TMR-DA was dissolved in 0.1 M Citric acid monohydrate–NaOH (pH 3.0) buffer. After injection the needle was kept in place for 10 min before slowly retracting it from the spinal cord. The wound was closed and the rats were kept for 4 –5 days before neuron culture or/and immunohystochemistry. Immunohystochemistry Four to five days were allowed for transport of the tracers, at which time the rats were deeply anesthetized with Metofane and perfused via the heart using 500 ml of 4% paraformaldehyde in PBS (pH 7.4). The brain was immediately removed and put in 4% paraformaldehyde 24 h followed by 30% sucrose solution.

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FIG. 1. Histofluorescent images of retrogradely labeled corticospinal neurons in cerebral cortex. TMR-DA was injected into the cervical (C1–C3) region of spinal cord, and the frontal cortex was processed for histofluorescence microsopy 4 days postinjection. In a (10⫻), note the TMR-DA fluorescing cell bodies in layer four of the frontal cortex. (b) A higher magnification of the same layer 4 (20⫻).

After the brain was immediately stored until sectioning. Fifty-micrometer transverse sections were cut using a freezing microtome and then mounted on slides. Sections were examined using a Nikon fluorescence microscope with an appropriate filters for different tracers. Corticospinal Neuron Culture Rats were anaesthetized with metofane, decapitated and the brain was placed in ice-cold solution of HibernateA, 2% B27 supplement, and 0.5 mM glutamine. After the meninges removed, the cortical areas of origin of corticospinal tract axons was dissected (1). After that the white matter as possible had been also removed. The further procedure for culturing the neurons we used a recently published method (2) with minor modifications. After plating, 10 ng/ml of FGF2 was added. Immunocytochemistry The retrograde labeling with TMR-DA was more clearly demonstrated by enhancing the signal, using anti-TMR-DA antibodies. Cells for staining with antiTMR-DA and/or anti-neurofilament 200 (NF200) antibodies were rinsed free of medium with PBS and fixed for 20 min at room temperature with 4% paraformaldehyde in PBS. After rinsing with PBS, cells were permeabilized for 10 min with 0.5% Triton X-100 in PBS. Nonspecific binding was prevented with blocking buffer (1% normal goat serum and 1% BSA in PBS). Than cells were incubated for 1 h at room temperature with rabbit anti-TMR-DA (Molecular Probes, A-6397, dilution 1 ␮g/ml) or mouse anti-NF200 (Sigma N5389, diluted 1:40) in PBS with 1% BSA. For double immu-

nostaining antibodies to NF200 and BrdU were added simultaneously. After three washes in PBS, appropriate secondary antibodies were incubated for 1 h: goat anti-mouse IgG FITC Conjugated (Sigma, F2012, dilution 1:64); goat anti-rabbit IgG Alexa 546 conjugated (Molecular Probes, A11010, dilution 1:500). After rinsing five time in PBS slips were mounted in Prolong Antifade kite (Molecular Probes, P-7481) and images analysed by microscopy. Controls omitting the primary antibodies or with normal rabbit serum were nonreactive. Incorporation of BrdU and Double Immunostaining with Anti-Brdu and Anti-TMR-DA Proliferating cells in vitro were detected by Brdu (Sigma, B5002) incorporation after incubation with 10 ␮M Brdu in growth medium for 24 h. Cells on coverslips were rinsed with PBS and fixed in 4% paraformaldehyde in PBS. After washing, cell DNA was denaturated by immersing coverslips in 2 N hydrochloric acid for 30 min. The coverslips then washed extensively in medium to restore a neutral pH. Monoclonal antibody against BrdU (Sigma, Clone BU 33) (1:1000 in blocking buffer) was used to detect incorporated BrdU. For double immunostaining the cells were incubated for 1 h at room temperature with mouse anti-BrdU and rabbit anti-TMR-DA. After rinsing with PBS, appropriate secondery antibodies were incubated for 1 h: goat anti-rabbit IgG Alexa 546 conjugated and goat antimouse IgG FITC conjugated. Glass coverslips were then mounted in ProLong Antifade reagent (molecular Probes) to retard fluorescence quenching and dried on microscope slides.

PROLIFERATION OF ADULT CORTICOSPINAL NEURONS

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FIG. 2. Phase contrast and BrdU immunostaining of retrogradely labeled cells with tetramethylrhodamine (TMR-DA) after 4 h, 1 day, and 2 days in culture. The TMR-DA fluorescence was enhanced using anti TMR-DA followed by secondary antibodies conjugated to Alexa 546.

Microscopy and Image Analysis Digitized immunofluorescence images were captured with a chilled CCD camera (PXL; Photometrics, Inc., Tucson, Ar) on Nikon Diaphot microscope equipped with a 10, 20, 40 oil/or not immersion objective and analyzed with Metamorph softwere (Universal Imaging, Corp., West Chester, PA). Statistical Analysis of Results Mean cell counts and standard errors of the mean have been presented within the text. Student’s t test was used to detect significant differences between means; P ⱕ 0.05 was considered significant. RESULTS AND DISCUSSION

To study the biology specifically of adult corticospinal tract neurons, we prelabeled this population by

axonal retrograde transport with fluorescence molecules in vivo before harvesting the cells, and this enabled us to follow these individually labeled cells in culture. A variety of tracer molecules were tested, including dextran tetramethylrhodamine (TMR-DA), dextran fluorescein (fluoro-emerald), dextran fluorescein and biotin, Oregon green, and fast blue, by making injections into the upper cervical spinal cord. With all tracers, the histofluorescence richly labeled the corticospinal tract neurons in layer 4 of the frontal cortex. The results showed that retrograde transport of TMR-DA was more efficient and the results were more constant than that of other tracers (Fig. 1). After dissociation and culturing of the labeled cells, however, the retrograde label faded for all the molecular tracers tested. This problem was overcome by optimizing a variety of variables including the pH of injection vehicle, sizes of the dextrans conjugated to the fluorescent molecules, and most importantly the amplification of

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FIG. 3. Phase contrast and double immunostaining with anti-TMR-DA and anti-BrdU of TMR-DA retrogradely labeled cells after 3 days in culture.

the fluorescent signal by using antibodies to tracer moleciles. When a weakly acidic or neutral injection buffer was used, the results varied from experiment to experiment. In contrast, with a strong acidic buffer more constant results were obtained in repeated experiments. Our optimized system uses 3 or 10 K dextranconjugated tetramethylrhodamine (TMR-DA) in a pH 3 buffer, and the amplification of the fluorescence for long-term cultures was accomplished by using antibodies to TMR-DA (after fixation in 4% paraformaldehyde), followed by secondary antibodies conjugated with Alexa 546. This amplification system theoretically enhances the signal 20⫻. It is these TMR-DA-labeled

cells along with all the other cells of the frontal cortex that were dissociated and prepared for cell culture. The cells were collected from the region of the brain containing neurons of the CST and dissociated in Hibernate A solution. About 15,000 cells/mg of tissue were obtained. After gradient purification, the cells were plated in the culture dish at about 350 cells/mm 2 in neurobasal A and B-27 media. Four hours after plating, 25% (89 ⫾ 2.4 cells/mm 2) of the cells attached and survived. In the presence of 10 ng/ml bFGF, this number was maintained. On days 3–5 there was a progressive increase in the total cell number. It doubled on the third day (184 ⫾ 9.65 cells/mm 2) and

FIG. 5. Phase contrast and double immunostaining with anti-TMR-DA and anti-NF200 of TMR-DA retrogradely labeled cells after 5 days in culture.

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FIG. 4. Phase contrast and double immunostaining with anti-TMR-DA and anti-BrdU of TMR-DA retrogradely labeled cells after 6 and 9 days in culture.

reached around 535 ⫾ 7.11 cells/mm 2 on the 5th day. On days 9 –10, the cell number plateaued, at 1556 ⫾ 15.9 cells/mm 2. Due to variations between preparations, including plating efficiency and labeling of cells in vivo, the results from different experiments were not combined. All experiments were repeated for qualitative confirmation. For quantitative analysis, (total cell number or/and TRM-DA-labeled cells) cells were counted through a photomask (calibration plate for cell counting) of an area of 0.125 mm 2 with a 20x objective. Eighty contiguous fields in each of two culture wells were counted at 4 h and at 1, 3, 5, 6, 9 days. For all microscopy and image analysis, digitized immunofluorescence images were captured with a chilled CCD camera on a Nikon Diaphot microscope equipped with a 20⫻ objective and analyzed with Metamorph software. Images of TMR-DA, BrdU, and NF200 were corrected for local background intensities. For NF-200 immunostaining, immages were corrected also for the

slight “nonspecific” immunofluorescence staining due to secondary antibody alone. For BrdU and TMR-DA the images were also corrected by subtracting the average slight nonspecific staining due to first and secondary antibodies of cells that were not treated with BrdU and not labeled in vivo with TMR-DA before harvesting the cells. The results show that adult corticospinal tract neurons survive, proliferate, regenerate processes, and establish neuritic networks in culture (Figs. 2– 4). That these proliferating neurons are in fact derived from adult neurons was demonstrated by fluorescent signal enhancement in neurons that were retrogradely labeled in vivo. At 4 h, about 1% of the total numbers of cells (0.775 ⫾ 0.085 cells/mm 2) were TMR labeled and appear quite isolated (Fig. 2a). At 1 and 2 days there was not any significant changes of the total number of cells and still there were quite isolated brightly TMR labeled cells (Figs. 2b and 2c). At 3 days the total number of cells had doubled (from 89 ⫾ 2.4 cells/mm 2

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to 184 ⫾ 9.65 cells/mm 2), as did the TMR-DA-labeled cells (from 0.775 ⫾ 0.085 cells/mm 2 to 1.5 ⫾ 0.247 cells/mm 2), P ⱕ 0.05. This suggested that TMR labeled cells have also doubled. At that time clusters of labeled cells with dividing cell morphology was characteristic (Figs. 3a and 3b). It was not possible to obtain counts of labeled cells beyond 3 days because of further fading of the retrograde label below our ability to detect with amplification methods. The reason for this could be the dilution of the dye with cell proliferation (Figs. 4b and 4c) or the death and detachment of labeled cells. However, even after 3 days and till 8 –9 days when a stable neuronal network was formed, there were few very bright TMR-DA-labeled cells, which are likely to be labeled cells that survive and either cannot or have not yet begun to proliferate (Fig. 4a). To further confirm that the labeled neurons undergo proliferation we tested if these retrogradely labeled cells synthesize DNA by adding 10 ␮M Brdu to the cultures 24 h prior to fixation of the cells (with the exception of the 4 h cultures where BrdU was added after plating the cells). After fixation in 4% paraformaldehyde in PBS and denaturation of cells DNA, a monoclonal antibody against BrdU followed by secondary antibody conjugated with FITC was used to detect incorporated BrdU. For double immunostaining, cultures were immunoreacted for BrdU and TMR-DA simultaneously. Four hours following plating of the cells in the presence of BrdU, no BrdU incorporation was observed (Fig. 2a). After 24 h in the presence of BrdU, about 15% (12.9 ⫾ 2.9 cells/mm 2) of the cells were BrdU positive, and a few were also TMR-DA labeled. At 3 days some of these double-labeled cells appear as dividing cells on the basis of morphology and BrdU immunoreactivity (Fig. 3a). In addition, clusters of TMR-DA labeled cells appear and some of them are BrdU immunopositive (Fig. 3b). At 9 days there were no longer any BrdU immunopositive cells. Some variation in the intensity of BrdU labeling is expected in nonsynchronized cultures because incorporation of label only occurs during S-phase. Double staining for TRM-DA and NF200 has shown that at 4 h, 1 day, and 2 days all TMR-DA labeled cells are NF200 immunopositive. At 5 days about 70% (364 ⫾ 12,1) of cells were NF200 positive. At that time the few cell that were found to be TMR-DA labeled were also NF200 immunopositive (Fig. 5). Thus adult corticospinal tract neurons in culture incorporate BrdU which show that they undergo DNA synthesis and counts of labeled cells with total cell

counts suggest further that at least a portion of these cells undergo cell division. Importantly, as with many groundbreaking works, there are a number of interesting issues that remain unanswered. Do these proliferating adult neurons, before entering the cell cycle, dedifferentiate to neuronal progenitors or multipotent stem cells? Do they give rise only to neurons or can they also differentiate into glial cells? Do these cells divided symetrically or asymetrically? And of fundamental importance, can these cells be induced to divide in vivo. ACKNOWLEDGMENTS The authors thank Mr. David Hobbs and Mr. Mattew Downey for technical assistance and helpful hints and suggestions and Dr. James R. Bamburg for critical reading of the manuscript. This work was supported by Spinal Cord Society.

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