Culture of neurons from postmortem rat brain

Neuroscience Letters 224 (1997) 193–196

Culture of neurons from postmortem rat brain Dennis Q. McManus a ,*, Gregory J. Brewer b a Department of Neurology, Southern Illinois University School Medicine, Springfield, IL 62794-1220, USA Department of Medical Microbiology and Immunology, Southern Illinois University School Medicine, Springfield, IL 62794-1220, USA

b

Received 12 February 1997; revised version received 24 February 1997; accepted 24 February 1997

Abstract Live neurons from pathological postmortem brains may provide a better model to study the molecular and cellular events associated with neurodegenerative disease. The aim of this study was to culture neurons from adult rat brain, 1 and 2 h postmortem, in typical normothermic autopsy conditions. We reliably cultured cells up to 2 h postmortem, in high yield, with neuron morphology, staining for neuronal markers (microtubule-associated protein 2, tau, and neurofilament 200). These neuron-like cells lacked glial marker staining (OX42 and glial fibrillary acidic protein). Our results suggest that neurons may be cultured from autopsy donors who have died either with or without a neurodegenerative disease such as Alzheimer or Parkinson disease.  1997 Elsevier Science Ireland Ltd. Keywords: Regeneration; Graft; Alzheimer; Parkinson; Stroke; Anoxia; Tau; Neurofilament

The cellular pathophysiology of age-related neurologic disease is poorly understood. Currently, cell culture of embryonic animal brain tissue is a popular method to explore the molecular and cellular events that may occur as a consequence of disease states, including cerebrovascular, Parkinson and Alzheimer disease. However, the developmental immaturity of embryonic cells limit application of their study to age-related disease. Without exposure to the aging process, it is difficult to determine the significance of embryonic cell culture findings to neurodegenerative disease. Also, significant moral concerns apply to the use of human fetal tissue. Until recently, there was no reliable method to isolate and culture neurons in significant numbers from mature brains. New techniques for culture of adult rat neurons now make it possible to examine the postmortem window of viability for adult neurons [6]. A major obstacle to using adult human tissue is the morbidity and mortality associated with brain biopsy. Another potential hurdle in the postmortem recovery of neurons, is the 5 min window for complete central nervous system (CNS) recovery after normothermic cardiac arrest without blood flow [15]. * Corresponding author. Tel.: +1 217 7858684; fax: +1 217 5244539; e-mail: [email protected]

However, some evidence suggests that individual neurons may survive beyond the point of clinical death. Neurons in culture can survive longer than 60 min under anoxia [10]. Viable neurons from brain cortex can be isolated several hours after death and even from frozen tissue [12]. There are several reports of successful culture of cells morphologically similar to neurons from human pre- and postmortem brains [1,2,7,9,16,18]. However, yields are low, and questions remain about their glial or neuronal identity. Still, with a postmortem interval under 2 h, it may be possible to reliably culture neurons from individuals with Alzheimer, Parkinson or cerebrovascular disease. With this in mind we determined, if neuron-like cells could be recovered from rats beyond the time constraints of anoxic brain death at typical room temperatures. Female adult Sprague–Dawley rats (250–300 g, n = 2) were anesthetized with phenobarbital (75 mg/kg) and sacrificed by decapitation in accordance with procedures approved by the animal care and use committee of our institution. After opening the skull and initial dissection, the unremoved brain tissue remained in the skull gradually cooling to room temperature (22°C). To avoid dehydration between samples, the skull was placed in a covered jar, humidified with normal saline. Frontal cortex samples (5– 15 mg) were resected by scalpel at 0, 1 and 2 h after sacri-

0304-3940/97/$17.00  1997 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940 (97 )0 0172-9

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fice. The 0 time point was actually 2–5 min postmortem, allowing time for decapitation and brain exposure. The tissue was placed in ice-cold medium (Hibernate A, 2% B27 supplement, 0.5 mM glutamine; GIBCO/Life Technologies, Gaithersburg, MD, USA) [4], the meninges and white matter were removed, and the tissue was chopped in 0.5 mm slices (MacIlwain, Brinkman, Wesbury, NY, USA). The cells were dissociated as described [6]. Briefly, tissue slices were digested with papain (2 mg/ml) for 30 min at 30°C in Hibernate A/B27. The tissue was triturated in three 2 ml volumes of Hibernate A/B27. The 6 ml of cell suspension was layered over a 4 ml step density gradient (Nycoprep at 15, 20, 25, and 35% in Hibernate A/B27; Life Technologies), and centrifuged 15 min, 800 g, at room temperature. The top 7 ml of supernatant was aspirated. The next 2.75 ml from the major band and below was collected and diluted in 5 ml of Hibernate A/B27. After centrifugation for 2 min, the cell pellet was resuspended in Neurobasal A/B27 (Life Technologies) and 0.5 mM glutamine, and plated at a concentration of 320 cells/mm2 onto 12 mm coverslips coated with 50 mg/ml poly-d-lysine (135 kDa; Sigma). Viable cells were determined by exclusion of 0.2% Trypan Blue

Fig. 1. Neuron-like cells from cortex, 0 and 2 h postmortem, after 5 days in culture show immunostaining for neurofilament and not GFAP. Cells were isolated from frontal cortex of an adult rat after 0 h (A–C) or 2 h postmortem (D–I). After 5 days in culture, cells were fixed and processed for double immunostaining with antibodies to neurofilament and GFAP. Phase contrast images are shown in (A), (D) and (G). Fluorescein fluorescence for neurofilament is shown in (B), (E) and (H). In (H), the anti-neurofilament antibody was omitted as a control. Rhodamine fluorescence for GFAP is shown in (C), (F) and (I). Arrow in (I) indicates a rare cell, weakly reactive for GFAP. Microglia (labeled mg) are clearly seen in each phase image. They show some immunoreactivity to neurofilament, perhaps because they have phagocytized debris containing neurofilament antigens. Scale bar, 10 mm.

Fig. 2. Significant yield of neuron-like cells remains after 5 days in culture for postmortem brain samples. Inset shows yield of isolated cells before culture. After 5 days in culture, neuron-like cells were counted in six adjacent fields/cover slip, two slips/animal, from two animals (mean, SE).

(Sigma). Cultures were incubated at 37°C with 5% CO2 and 9% O2 for 1 h. Coverslips were drained and rinsed with 0.4 ml Hibernate A to remove debris. The rinse was immediately replaced with 0.4 ml of Neurobasal A/B27, 0.5 mM glutamine, 5 ng/ml FGF2 (Life Technologies), 50 u/ml penicillin, 0.05 mcg/ml streptomycin and incubated as above. After 5 days of growth, viable cells were stained by active uptake of fluorescein diacetate and dead cells stained with propidium iodide [5]. Most cells were fixed for 15 min in phosphate-buffered 4% paraformaldehyde. For glial fibrillary acidic protein (GFAP) and neurofilament 200 (NF200) double immunostain, cells were fixed for 20 min in 10% acetic acid and 90% ethanol. After rinsing with phosphate-buffered saline (PBS), cells were incubated 5 min in PBS containing 0.5% Triton X-100, and 5% normal goat serum (NGS); except the galactocerebroside (GalC) stain used 1% bovine serum albumin (BSA) and 1% NGS without detergent, and the microtubule associated protein 2 (MAP2) and tau double stain used 1% BSA and 5% NGS with 0.5% Triton X-100. Cells were next incubated overnight at 4°C with primary antibody diluted in PBS containing 0.05% Triton X-100 (except GalC stain omitted Triton X-100). Primary antibodies were as follows: mouse monoclonal anti-NF200, 1:40 (Sigma); mouse anti-MAP2, 1:50 (Sigma); rabbit anti-tau, 1:20 (Sigma); rabbit anti-GFAP, 1:2000 (DAKO, Carpinteria, CA, USA); rabbit anti-GalC, 1:50 (Sigma); mouse monoclonal anti-OX 42 (OX42), 1:5 (Accurate, Westbury, NY, USA). After four rinses with PBS, the primary antibody was detected with appropriate secondary antibodies incubated for 1 h at room temperature diluted in PBS containing 0.05% Triton X-100 (except GalC stain omitted Triton X-100). After rinsing four times in PBS and mounting, the labeled cells were visualized using an epifluorescence inverted microscope (Nikon), 100 W mercury lamp excitation through a 0.5 neutral density filter, a 60 × /1.4 NA objective, B1A or G1B dichromic mirrors and recorded on Ektachrome ASA 800 film. Non-specific label-

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Fig. 3. Neuron-like cells from cortex, 2 h postmortem, after 5 days in culture show immunostaining for neuron cytoskeletal proteins MAP2 (B) and tau (C). Arrows indicate weak MAP2 immunoreactivity relative to stronger tau reactivity, suggestive of axons. Phase contrast images are shown in (A) and (D). In (E) and (F), the anti-MAP2 and anti-tau antibodies were omitted as a control. Scale bar, 10 mm.

ing was determined by incubation without primary antibody. Viable neuron-like cells were successfully cultured from specimens obtained at 0, 1 and 2 h postmortem. Cells cultured from the 2 h postmortem brain (Fig. 1D) appeared similar to freshly isolated neurons (Fig. 1A). We measured viable cells at the time of isolation by exclusion of trypan blue and after 5 days in culture by uptake of fluorescein diacetate. Isolated viable cells declined slowly with the postmortem interval (Fig. 2, inset). At 2 h postmortem, the total number of isolated viable cells (5600 cells/mg tissue) declined only 30% from fresh specimens (7900 cells/mg tissue). After 5 days in culture the fraction of cells with neuron-like morphology declined with postmortem interval. The yield of neurons from the 2 h postmortem 5 day culture dropped more than 50% compared to the 0 h time (Fig. 2). Table 1 provides an accounting of viable cells normalized to weight of processed tissue. Before culture, the yield of isolated cells at 2 h postmortem was 72% of the 0 h time (Fig. 2, inset). After 5 days in culture, the percentage of viable neuron-like cells at the 2 h postmortem collection was 30% of the 0 h time (Table 1; Fig. 2). Cells with neuronal morphologies under phase contrast microscopy were recognized by their reactivity with antibodies to neurofila-

ment 200 (Fig. 1B,E). Only rarely were any cells stained for GFAP (Fig. 1C,F,I). Microglia were the other major cell type in these cultures (Fig. 1A,D,G). They are large, flat cells that stain with antibodies to OX42 (data not shown). In addition to morphological and neurofilament staining criteria, neuron-like cells were also immunoreactive with antibodies to two other markers of the neuron cytoskeleton, MAP2, and tau (Fig. 3B,C). After 5 days of regeneration in culture, MAP2 and tau appear to co-segregate. However, there was some enhanced staining for tau over MAP2 in the finer neurites that could be axons (Fig. 3C, arrows). We have demonstrated that a significant number of neuron-like cells can be obtained up to 2 h postmortem. The high yield, their neuron-like morphologies, staining for neuronal markers and lack of staining for glial markers suggest that they are actually neurons. Our successful culture of neuron-like cells at the 1 and 2 h collection times is two orders of magnitude greater than reported for the culture of colonies of cortical neuronal progenitors [13]. After 2 h of postmortem ischemia, a significant fraction of neurons still can be rescued from cell death. Lastly, we found tau immunoreactivity in our cultures that were allowed to regenerate for only 5 days without passage. Tau immunoreactivity was not seen in neuronal progenitor cultures allowed multiple passages to differentiate [8]. Even if the cells we isolated are neuronal progenitors, any reliable postmortem yield would remain useful for the study of age-related neurologic disease. The reasons for the improved yield and postmortem regeneration of the neuron-like cells needs further study. The Neurobasal/B27 medium may be part of the explanation. During isolation of cells from the brain tissue, the pH was controlled without bicarbonate buffering [4]. Neurobasal A is a base medium of higher osmolarity than Neurobasal [6]. Neurobasal is modified from Dulbecco’s modified Eagle’s medium (DMEM) with the addition of vitamin B12 and ZnSO4 from F12 medium, but without the FeSO4 and glutamate, which are toxic to neurons [5]. B27 contains the five ingredients in N2 (insulin, transferrin, selenium, putrescine, progesterone), two other hormones, corticosterone and T3, and 13 other nutrients [5]. Among the other nutrients are essential fatty acids and ethanolamine that may facilitate membrane synthesis for neurite outgrowth and five antiox-

Table 1 Viable yield of cells depends on the postmortem interval Postmortem interval

0h 1h 2h

Cells isolated per mg cortex

Cells after 5 days culture per mg cortex

Total cells (% of reported 170K/mg cortex) [3]

All viable cells (% of cells isolated/mg)

Viable neuron-like cells (% of all viable cells/mg)

7692 (4.5) 5161 (3.0) 5576 (3.3)

1394 (18) 605 (12) 1194 (21)

978 (70) 165 (27) 293 (25)

All cell counts are based on original wet weight of frontal cortex dissected. Comparisons are made to reported cortical neuron density of 170K/mm3 in mouse

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idants (vitamin E, vitamin E acetate, glutathione, catalase and superoxide dismutase). The antioxidants are protective against the reactive oxygen species that are generated during experimental ischemia [11]. After selecting theoretically beneficial components of B27, each was optimized for embryonic hippocampal neuron survival after 4 days in culture. These factors evidently contributed to survival of adult neurons [6]. Our current efforts center around the subtypes of cells that survive, and if there is selective time-dependent viability. Other work in rats indicates that ischemia is selectively toxic to striatal, hippocampal and cortical neurons [14], and that GABAergic neurons are selectively preserved [17]. There is a need to further study the endurance, electrophysiology, ultrastructure and molecular biology of cells cultured by the above method.

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Supported by Life Technologies, Alzheimer’s Association and Mr. and Mrs. Malan’s research gift. We are indebted to Mr. John R. Torricelli and Mr. John Viel for technical assistance. We thank Dr. Dean K. Naritoku for critical reading of the manuscript.

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