ngf-mediated synaptic sprouting in the cerebral cortex of lesioned primate brain

BRAIN RESEARCH

ELSEVIER Brain Research 692 (1995) 154-160

Research report

NGF-mediated synaptic sprouting in the cerebral cortex of lesioned primate brain

Ivan Burgos a, A. Claudio Cuello b, Paolo Liberini b, Eric Pioro b, Eliezer Masliah a, * a Department of Neurosciences, University of California, San Diego, School of Medicine, La Jolla, CA 92093-0624, USA

b Department of Pharmacology and Therapeutics, McGill University, Faculty of Medicine, 3655 Drummond, Montreal, Que. H3G 1}I6, Canada

Accepted 16 May 1995

Abstract

In the present study, coronal brain sections of cortically devascularized non-human primates (Cercopithecus aethiops) were used to assess the lesion-associated synaptic loss, and the effect of exogenous nerve growth factor (NGF) in preventing or reversing this neurodegeneration. The sections were immunolabeled with antibodies against the synaptic marker protein synaptophysin (SYN), as well as choline acetyltransferase (CHAT) and parvalbumin (PV) markers that identify cholinergic neurons and interneurons, respectively. We found that, compared to sham-operated animals, in the lesioned vehicle treated animals SYN immunoreactivity near the lesioned site in the frontoparietal cortex was decreased by 31%. Similarly, corrected optical density values of immunostained sections specific for ChAT in the nucleus basalis of Meynert (ipsilateral to the lesion) decreased by 20% and PV-immunoreactive neurons near the lesion decreased by 47%. In contrast, NGF-treated lesioned animals showed levels of SYN, CHAT, and PV immunoreactivity similar to sham controls. These results are consistent with previous studies and support the view that NGF may not only prevent neurodegenerative changes after neocortical infarction by protecting vulnerable neurons, but also is capable of inducing sprouting and synaptogenesis.

Keywords: Nerve growth factor; Choline acetyltransferase; Parvalbumin; Nucleus basalis of Meynert; Cholinergic neuron; Cholinergic synapse

1. Introduction

Repairing and restoring function of injured neural path- ways is the ultimate goal of regeneration studies in the central nervous system (CNS). This goal requires that some degree of reorganization in the neural circuitry and connections should occur. Potential candidates to mediate these phenomena are trophic factors of which nerve growth factor (NGF) is a prototype. Numerous studies have demonstrated the neurotrophic effects of NGF on basal forebrain cholinergic neurons in vivo [2,5,8,30,31]. How- ever, despite these studies little is known about the mecha- nisms by which NGF promotes cholinergic synapse remod- eling in the injured mammalian brain [9].

Only limited experimental work has been carried out in primate experimental models of neurodegeneration that more closely correlate with the alterations observed in pathological human brain. In this regard, recent studies in

* Corresponding author. Fax: (1) (619) 534-6232.

0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0006-8993(95)00696-6

non-human primates (Cercopi thecus aethiops) have shown that after cortical infarction by meningeal devascularization, neuronal cell bodies in the intermediate nucleus basalis of Meynert (NbM) shrink and neuritic processes are lost [15,16,23]. It has also been reported that choline acetyltransferase (CHAT) activity decreased in the NbM most likely as a result of retrograde degeneration, and in the areas surrounding the neocortical infarction either as a result of a global retraction of cholinergic network [9], or the anterograde degeneration of fibers passing through the lesioned site [16]. In this experimental primate model, administration of NGF fully prevented the retraction of cholinergic neurons and partially reversed the loss of CHAT-positive neuritic meshwork in the cortex caused by the lesion [16]. On this basis, we hypothesized that neuroprotective effects elicited by NGF in the cerebral cortex of primates bearing cortical infarction, as is the case in rodents, are associated with an increase in pre-synaptic elements. To test this hypothesis, we investigated the immunoreactivity (IR) of the vesicle protein synaptophysin (SYN), as an indicator of the number of presynaptic terminals in this experimental setting.

L Burgos et al./Brain Research 692 (1995) 154-160 155

2. Materials and methods

2.1. Experimental design

Six adult male monkey (Cercopithecus aethiops; weight 4 -6 kg, age range 5 -9 years) were used for this study. Animal acquisition, husbandry and postoperative care were provided by Carribean Primates, Ltd. (St. Kitts, EC). Facil- ities, surgical procedures and perioperative handling were in accordance with the McGill University Animal Care Regulations and the requirements of the Canadian Council on Animal Care. After random division into 3 groups: (1) sham-operated (n = 2), (2) lesioned vehicle treated (n = 2), and (3) lesioned recombinant human NGF (rhNGF)-treated (n = 2), the animals underwent a craniotomy, cortical devascularization and rhNGF treatment, as previously described [15]. Following surgery, animals were housed in individual cages and monitored daily for neurological deficits and signs of distress. They had free access to water and were regularly fed with fruit and High Protein Monkey Chow (Purina Mills, Inc.). Six months after surgery, the animals were intracardially perfused with fixative and the brains processed for immunocytochemistry.

2.2. Surgical procedures

Surgery and rhNGF preparation have been previously described [15,16,23]. Briefly, after induction of general anesthesia with an intramuscular injection (i.m.) of a mix- ture of ketamine sulfate (Ketaset, 15-20 mg/kg; Austin, Que.) and xylazine (Rompun, 1.5-2.0 mg/kg; Bayvet, Ont.) the head was secured in a Kopf stereotaxic head frame and a wide inverted U-shaped incision was made in the scalp on either side of the left ear. The underlying temporal muscle was reflected inferiorly and a 3.5 cm X 5.0 cm craniotomy was made with a dental drill. The posterior frontal, superior temporal and parietal cortices were exposed. In lesioned animals, pial blood vessels supplying the gyri in these regions were coagulated using a Malis bipolar cautery device (Codman, MA). Cortical vessels of sham-operated monkeys were not injured. A gelatin film incorporating either 2.8 mg of rhNGF or vehicle, was placed onto the exposed area of neocortex and a covering piece of sterile Gelfoam (Upjohn, Kalamazoo) secured it in place. After suturing of temporalis muscle into place, the skin was closed with interrupted sutures. The monkeys received a 1-week course of daily penicillin G i.m. (40,000 U/kg).

2.3. Tissue preparation

After the monkeys were deeply anesthetized with ketamine HC1 (20 mg/kg), the descending aorta was clamped and systemic vasculature was flushed through the ascending aorta with 500 cc of phosphate buffer (PB, pH 7.4). The solution was then changed to buffered 4% formalde-

hyde-0.05% glutaraldehyde/0.1 M in PB, with 1000 cc infused over 30 min. Brains were immediately removed, put into 10% sucrose-PB and stored at 4°C for up to 2 weeks. After blocking the brain to include the entire lesioned area, the hemispheres were sagitally divided and sectioned on a sledge microtome equipped with a freezing stage (Baldwin, Inc., Cambridge, UK). Fifty /xm-thick sections were serially collected in phosphate-buffered saline (PBS, pH 7.4) and stored free floating at 4°C until the antibody incubations.

2.4. Immunocytochemical analysis

Synaptophysin-IR was used to assess the overall changes in synaptic populations [18]. Further assessment of retrograde cholinergic degeneration was performed by ChAT immunocytochemistry [16]. Additional immunocytochemical analysis of parvalbumin (PV)-immunoreactive neurons was performed in order to study the alterations in interneurons [19]. Briefly, as previously described [18], sections were pretreated at room temperature with 3% hydrogen peroxide and 0.1% Triton X-100 in PBS (pH 7.4) for 20 min. The sections were then washed twice (10 min each) in PBS, placed in SuperBlock (ScyTek Labora- tories, logan, UT) for 7 min and rinsed quickly with PBS. For SYN immunocytochemistry, sections were incubated overnight at 4°C in a mouse monoclonal antibody (SY38, 1:10, Boehringer-Mannheim, Indianapolis, IN), which is reactive against the synaptic protein marker synaptophysin [12,21,29]. On the following day, the sections were incubated for one hour at room temperature in fluorescein-con- jugated horse anti-mouse IgG (Vector Laboratories, Burlingame, CA). Experimental studies in animal models of denervation and reinnervation have shown that SYN is a good and sensitive marker for presynaptic terminals [18].

For ChAT immunocytochemistry, the sections were washed twice (10 min each) and after pretreatment and blocking incubated overnight at 4°C in rabbit anti-ChAT primary antibody (AB143, 1:500, Chemicon, Temecula, CA). The following day, the sections were incubated for one hour in biotinylated goat anti-rabbit secondary antibody (1:100, Vector). For PV immunocytochemistry, sections were incubated overnight in mouse monoclonal anti- PV antibody (PA2350, 1:750, Sigma Chemical Company, St. Louis, MO), followed by a l-h incubation in biotinylated horse anti-mouse secondary antibody (1:75, Vector). After the incubation in the secondary antibodies, both sets of sections were incubated for l h at room temperature in the Avidin-HRP complex (1:100, ABC Elite, Vector). Each set of sections was then incubated for 5 min in diaminobenzidine tetrahydrochloride containing 0.03% hydrogen peroxide, mounted onto slides and coverslipped. All sections were immunostained simultaneously and under the same conditions. Sets of two sections from each group were immunolabeled in order to assess the repro- ducibility of the results.

156 1. Burgos et al. / Brain Research 692 (1995) 154-160

2.5. Morphometric analysis and statistics

Blind-coded SYN immunostained sections were viewed with a Zeiss 63 x (N.A. 1.4) objective on a Zeiss Axiovert 35 microscope equipped with a laser scanning confocal system (MRC600, BioRad, Wattford, UK) [17]. Three fields from the frontoparietal cortex surrounding the lesioned area and one field from the temporal cortex and insula away from the lesion were examined. All sections from each experimental group were digitized under stan- dardized conditions, maintaining the same levels of gain, aperture and black. Quantitative analysis of the optical section images displaying the presynaptic terminals were carried out as previously described [17,18] with the aid of the Image 1.23 software program running on a Macintosh Ilci. The results were expressed as presynaptic terminals per 100 /zm:.

Parvalbumin-IR was assessesed in the frontoparietal cortex 'near the lesioned area, the temporal cortex, the globus pallidus and the putamen by quantification with a light microscope (10 × ocular, 40 × objective, field size = 0.16 /zme). Visual quantification of twelve fields from the frontoparietal cortex and of three fields from each of the other brain regions was carried out. For each immunos-

tained section, the results were expressed as mean number of immunostained interneurons per 0.16 /xm:. Sections immunostained with anti-ChAT were analyzed with the Quantimet 570C (Leica) as previously described in order to estimate the optical density of immunocytochemical reaction [20]. Corrected optical density (COD) was ob- tained by subtracting the white matter optical density. After the morphometric study was completed the code was broken for analysis and statistical differences among the three groups were analyzed by one factor ANOVA. All results were expressed as mean + S.E.M.

3. Results

Immunocytochemical analysis of tissue sections ob- tained from the untreated, neocortically devascularized C. aethiops showed that SYN-IR in the areas surrounding the lesioned site, decreased significantly by 31% when compared to sham-operated animals (63.9 + 5 vs. 92.3 + 1 synapses per 100 /zm 2, Fig. 1A and 2). However, no statistically significant differences were noted in tissue sections from lesioned rhNGF-treated animals (87.1 + 4 synapses per 100 /xme), compared to sham-operated con-

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Fig. 1. Analysis of synaptophysin-immunoreactive nerve terminals showed that: (A) compared to sham operated animals, lesioned, untreated animals displayed a significant 31% decrease in SYN-immunoreactivity in the areas adjacent to the lesioned site. No statistically significant differences were found when comparing lesioned rhNGF-treated animals to sham operated controls. Analysis of regions away from the lesion site such as frontoparietal cortex (B), the temporal cortex (C), and insula (D) and showed no differences in SYN-immunoreactivity among the three groups.

L Burgos et al./Brain Research 692 (1995) 154-160 157

Fig. 2. Patters of SYN-immunoreactivity in control and lesioned cortex imaged with the laser scanning confocal microscope, a: a cortical field of frontoparietal cortex. Sham operated animals displaying the characteristic punctate patterns of SYN-immunoreactivity in the neuropil, b: the frontoparietal cortex of a lesioned animal treated with rhNGF showing a similar patterns to that represented in a. c and d illustrate the marked decreasc in SYN-immunoreactive presynaptic terminals observed in the frontoparietal cortex adjacent to lesions. Magn. × 795.

trols (Fig. 1A and 2). Furthermore, analysis of regions away from the lesion such as in the temporal cortex, insula and non-lesioned frontoparietal cortex showed that SYN-IR was not different among the three groups (Fig. 1B-D). Microscopic representations of variations in the density of cortical SYN immunoreactive elements are shown in Fig.

Choline acetyl transferase-IR in lesioned vehicle-treated

animals was decreased significantly by nearly 20% in the NbM, when compared to sham-operated animals (Fig. 3A). In contrast, ChAT-IR in the NbM was higher by approxi- mately 20% in the lesioned rhNGF-treated animals when compared to sham-operated animals (7.9 + 0.1 vs. 6.4 + 0.3 COD), and 37% higher compared to lesioned vehicle- treated animals (Fig. 3A).

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158 L Burgos et aL / Brain Research 692 (1995) 154-160

ing the lesion was significantly decreased by 47% when compared to sham-operated animals (Fig. 3B). The mean number of PV-immunoreactive neurons was 12 + 1 for the lesioned untreated group and 23 + 1 for the sham-operated animals (Fig. 3B). In contrast, a complete reversal of the effects of the lesion was observed in the lesioned rhNGF- treated animals, as demonstrated by a lack of significant difference in the mean number of PV-immunoreactive neurons between the rhNGF-treated and sham-operated groups (21 + 1 vs. 23 + 1) (Fig. 3B). In addition, the mean number of neurons in the temporal cortex, globus pallidus and putamen away from the lesion was not significantly different between the sham-operated, lesioned vehicle- treated and lesioned rhNGF-treated groups (Fig. 3B).

4. Discussion

4.1. Effects o f cortical devascularization

In the present study the effects of unilateral cortical devascularization in C. aethiops was assessed by immuno- cytochemicai techniques specific for presynaptic boutons, independently of the nature of the transmitter utilized. This procedure does not distinguish boutons from projecting or local circuit neurons. We found that the density of both presynaptic terminals and PV-immunoreactive neurons, after cortical devascularization, was significantly reduced in the cortical areas surrounding the lesion, while in regions away from the lesion it was unchanged. The decreased density of SYN-immunoreactive presynaptic terminals is consistent with previous reports showing shrinkage of cortical cholinergic boutons and loss of synaptic contacts associated with a decrease of ChAT-IR in the cortical fiber network after unilateral devascularizing cortical lesions [8,9,16].

While in our study the ischemic lesion was associated with a significant decrease of PV-immunoreactive neurons, previous studies have shown preservation of this neuronal population in the striatonigral system of rats after transient unilateral middle cerebral artery occlusion (MCAO) [10] and have also demonstrated preservation of PV-immunoreactive neurons in the neocortex and hippocampus of mongolian gerbils after transient forebrain ischemia via bilateral clipping of the carotids [27]. However, it should be emphasized that the disparity between these studies and our observations can be attributed to the difference in the lesion models. It may be that permanent cortical devascularizing lesions result in an interaction with tissue, as opposed to the ischemia are characterized by general irre- versible damage and cell loss of various populations of neurons, whereas the effects of temporary ischemia are associated with selective vulnerability of a restricted number of neurons. Furthermore, other studies have shown a delayed and reversible decrease of PV-immunoreactive neurons in the rat hippocampus after transient cerebral

ischemia, suggesting that time factors are also important variables to considered in the assessment of the density of PV-IR [13]. In this regard, it should be noted that in the present experimental model the animal survival time after lesioning was 6 months.

The mechanism of injury responsible for the observed reduction in presynaptic terminals near the lesion site may also be associated with the anterograde degeneration of axonal fibers passing through the lesion site [16]. How- ever, the major component of the overall loss of presynaptic elements is the global retraction of the baso-cortical neurons and the loss of cortico-cortical projections. It has been repeatedly demonstrated that these devascularizing lesions provoke a retrograde degeneration of neurons in the nucleus basalis magnocellularis of rats and in the NbM of monkeys [2,16]. Consistent with these studies, we found a loss of cholinergic neuronal optical density in the NbM as shown by the significantly decreased ChAT-IR in this region. Neocortical infarction results in a loss of target sites and of cholinergic terminal networks within the in- farcted area and this, in turn, leads to a disruption of the pathway between the NbM and the rest of the cortex [9,16]. Consequently, the cholinergic fibers ascending to the cortex from the nucleus basalis degenerate gradually in a retrograde fashion provoking the well documented neuronal shrinkage and loss of neuronal processes [9], which explains the decreased biochemical ChAT activity in the NbM reported in both rat and monkey animal models [25]. In conclusion, the present experimental model does not entirely reproduce the neuropathological features of chronic neurodegenerative diseases; however, it represents an at- tractive approach to study both processes of cortical synaptic damage and NbM retrograde degeneration following cortical infarction.

4.2. Response to N G F administration

The present study showed that human recombinant NGF administered for a brief period of time can induce an almost complete recovery of SYN-immunoreactive synaptic populations and PV-immunoreactive neurons in the neocortex, and also induce preservation of cholinergic neuron phenotype in the NbM six months after ischemic decortication. From this investigation it is unclear whether this sprouting response arises from injured fibers or from intact axon collateral. However, these findings suggest that exogenous NGF may aid injured neurons in reestablishing connectivity. Such an occurrence might account for the preservation of NbM neuronal morphology in our experimental animals, even about 5 months after discontinuation of treatment [16]. These results are consistent with previous studies demonstrating that exogenous administration of NGF is capable of inducing neuronal sprouting [1,3,6,7,9] and synaptogenesis [1,9]. It has been shown that cortical cholinergic fiber length, cortical ChAT-immunoreactive varicosities, and the actual number of ChAT-immuno-

L Burgos et al./Brain Research 692 (1995) 154-160 15t~

reactive synapses are increased in rats with unilateral, cortical devascularizing lesions after 7 days of NGF administration via minipumps [9]. In addition to these anterograde trophic activities of NGF, the neuroprotective activity of NGF against the retrograde degeneration of the basal forebrain cholinergic neurons has been extensively demonstrated in both rodents [5,11,24,26,31] and primates [14,16,28].

Another mechanism by which NGF administration causes the effects observed may be explained in terms of the ability of NGF to prevent neurodegeneration and trophic changes after cortical infarction. It has been suggested that growth factors may protect neurons against ischemic damage by stabilizing calcium homeostasis [22]. Studies have also shown that neuronal death following an ischemic event is preceded by a pathological accumulation of intracellular calcium. This elevated intracellular calcium acti- vates biochemical processes which cause enzymatic break- down of proteins and lipids, dysfunction of the mito- chondria, energy deprivation and eventually cell death [4]. Taken together, these studies would support the possibility that, in addition to its prototypical neurotrophic actions, NGF might ameliorate the neuronal damage caused by ischemic lesions, facilitating aspects of the synaptic re- modeling of affected CNS areas. Other possibilities imply broader actions of NGF yet to be studied.

Acknowledgements

The authors would like to thank Mrs. Margaret Mallory for her expert technical assistance. This work was sup- ported by NIH Grants AG10869 and AG05131 (E.M.) and a grant from the Medical Research Council of Canada (A.C.C.).

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ngf-mediated synaptic sprouting in the cerebral cortex of lesioned primate brain

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