Neocortical infarction in subhuman primates leads to restricted morphological damage of the cholinergic neurons in the nucleus basalis of Meynert

BRAIN RESEARCH ELSEVIER

Brain Research 648 (1994) 1-8

Research Report

Neocortical infarction in subhuman primates leads to restricted morphological damage of the cholinergic neurons in the nucleus basalis of Meynert Paolo Liberini, Erik P. Pioro, Dusica Maysinger, A. Claudio Cuello * Department of Pharmacology and Therapeutics, McGill University, Mclntyre Medical Sciences Building, 3655 Drummond Street, Montreal, Que., H3G 1}'6, Canada Accepted 22 February 1994

Abstract

The aim of the present study was to investigate the long-term effect of cortical infarction on the subhuman primate

(Cercopithecus aethiops) basal forebrain. The lesion, carried out by cauterizing the piat blood vessels supplying the left fronto-parieto-temporal neocortex, induced retrograde degenerative processes within the ipsilateral nucleus basalis of Meynert. The morphometrical analysis revealed that significant shrinkage of cholinergic neurons and loss of neuritic processes were localized within the intermediate regions of the nucleus basalis. The average cross-sectional areas of choline acetyltransferaseimmunoreactive neurons in the intermedio-ventrai (Ch4iv) and intermedio-dorsal (Ch4id) nucleus basalis were decreased to 62.5 + 9.5 and 58.0 + 8.6%, respectively, of the sham-operated values. Although an apparent loss of Nissl-stained magnocellular neurons in Ch4iv and Ch4id was found by applying a quantitative analysis based on a perikaryal-size criterion, data obtained by the quantification of immunostained material failed to reveal any significant decrease of cholinergic cell density. Results are discussed in view of future application of this ischemic model to study processes of retrograde degeneration following cortical target removal and to assess potential neurotrophic and neuroprotective properties of pharmacologic agents.

Key words: Acetylcholine; Basal forebrain; Ischemia, Nerve growth factor; Neurodegeneration; Nucleus basalis of Meynert

1. Introduction

In the last decade, the cholinergic neurons of basal forebrain have received remarkable experimental attention because of the degeneration which occurs in Alzheimer's disease (AD) [2,50,57] as well as in a variety of other pathologies, such as Korsakoff's disease [2], Down's syndrome [32], progressive supranuclear palsy [51], ischemic brain diseases [19] and dementia pugilistica [55]. It has been indicated that the degeneration of these neurons and the accompanying loss of cholinergic projections to various cortical and subcortical areas is directly related to the emerging cognitive impairment [3,6,10,20,25,39,43]. A marked reduction of choline acetyltransferase activity (CHAT) in the cerebral cortex and nucleus basalis of Meynert

(nbM) of patients affected by AD has been shown in classical studies [4,10,12,44]. The loss of ChAT activity detected in this disease is accompanied by neuropathological changes affecting the cholinergic neurons within the nbM and other regions of the basal forebrain complex [2,13,17,32,40,42,56-58]. Some of these studies have reported extensive neuronal loss of large-diameter cells within the nbM [2,17,32,41,57]. By contrast, other morphometric investigations have emphasized the prominence of cell atrophy and dysfunction rather than neuronal death [1,16,42,56,58]. It is remarkable that the severity of these neuropathological changes affecting the cholinergic neurons within the basal forebrain follows a regional distribution which correlates with the degenerative changes observed in the neocortex receiving the corresponding projections

[41]. * Corresponding author. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 4 ) 0 0 2 4 5 - 8

On this clinical basis, the study of the anatomical and functional organization of the basal forebrain in non-human primates has recently assumed critical im-

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P. Liberini et al./Brain Research 648 (1994) 1-8

portance in the effort to establish experimental models of neurodegeneration. Acetylcholinesterase histochemistry and ChAT immunocytochemistry have been extensively used to identify cell bodies and neuritic processes of forebrain cholinergic neurons [21,34-36]. The recent characterization of a monoclonal antibody directed against the low-affinity receptor for the nerve growth factor (NGF; p75 LNGw) has led to the demonstration that p75 LNGFR immunoreactivity is colocalized with ChAT immunoreactivity in monkey [26] and human forebrain neurons [27,40]. This monoclonal antibody has been adopted in many neuropathological studies for quantitative assessment of forebrain damage in AD [41] as well as in investigations assessing the NGF-neuroprotective activity in subhuman primates [23,24,39,53,54]. Using ChAT and p75 LNGFr~ immunocytochemistry, it has been shown that the human forebrain cytoarchitecture is analogous to that of other primates [34,37,40,41]. The Ch4 group is the largest forebrain region and can be divided into the same components that were identified in the monkey brain [34,40,41]. These anatomical and cytoarchitectural analogies prompted us to reproduce in non-human primates (Cercopithecus aethiops) the rodent cortical devascularizing lesion model [7,8,14,45,48]. Our aim was to study the cascade of retrograde degenerative processes affecting the nbM after neocortical infarction. In particular, we tried to define whether the morphological changes of the nucleus basalis consist of cell loss, phenotypical alterations or both. Therefore, we have visualized basal forebrain neurons using cholinergic immunocytochemical markers and performed a detailed morphometrical analysis of each nbM subregion.

(Rompun; Bayvet, Ont.; 1.5-2.0 mg/kg) mixture, the scalp was cleaned and shaved; anesthesia was maintained intraoperatively with intermittent i.m. administration of the same mixture (10-15 m g / k g / 3 0 rain). I.m. injection of dexamethasone (Austin, Que.; 0.2 mg/kg) and penicillin G (Ayerst, Montreal; 40,000 U / k g ) was given preoperatively. The monkey's head was secured in a Kopf stereotaxic head frame and the left hemicranium was washed with iodine and alcohol. Following periosteal infiltration with 2% xylocaine (Astra, Ont.) for greater analgesia, 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 3.5×5.0-cm craniotomy was made with a dental drill. The skull flap was elevated and the underlying dura mater was opened with an iridectomy microscissor and reflected inferiorly. This craniotomy consistently exposed a wide area of the left cerebral hemisphere including posterior frontal, superior temporal, parietal and anterior occipital cortices (Fig. 1). 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 and those of the primary motor cortex (Brodmann Area 4) were not disturbed in lesioned animals. Adequate obliteration of vessel lumina was evidenced by the resultant pallor of the subjacent cortical parenchyma. After repositioning the dura mater and suturing the temporalis muscle into place, the skin was closed with interrupted sutures. The monkeys received another i.m. injection of dexamethasone postoperatively (0.2 mg/kg) as well as a l-week course of daily penicillin G i.m. injections (40,000 U/kg). Motor and sensory deficits were documented and animals manifesting signs of distress were given i.m. analgesics. 2.3. Immunocytochemistry After the monkeys were deeply anesthetized with ketamine HCI (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% formaldehyde-0.05% glutaraldehyde/0.1M in PB (pH 7.4) with 1000 cc infused over 30 min. Brains were immediately removed, put into 10% sucrose-PB (pH 7.4) and stored at 4°C for up to 2 weeks. After blocking the brain to include the entire nbM [49], the hemispheres were sagitally divided and sectioned on a sledge microtome

2. Materials and methods 2.1. Animals Eight adult male C. aethiops (4-6 kg, 5-9 years) were used in this study. Animal acquisition, husbandry and postoperative care were provided by Caribbean Primates (St. Kitts, EC). Facilities, surgical procedures and perioperative handling were in conformity with the McGill University Animal Care Regulations and the requirements of the Canadian Council on Animal Care. After random division into two groups (four sham-operated, four lesioned), the animals underwent craniotomy and eventual cortical devascularization. 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). After 6 months' survival, the monkeys were intracardially perfused with fixatives and their brain were processed for immunocytochemistry. 2.2. Surgery After induction of general anesthesia with an i.m. injection of ketamine sulfate (Ketaset; Austin, Que.; 15-20 mg/kg) - xylazine

Fig. 1. Schematic representation of C. aethiops brain hemispheres showing localization and extent of devascularized region. In lesioned animals, after removing a fronto-parieto-temporal skull flap (3.5 × 5.0 cm), pial blood vessels supplying exposed neocortex were coagulated with a bipolar cautery device.

P. Liberini et al. / Brain Research 648 (1994) 1-8 equipped with a freezing stage (Baldwin, Cambridge, UK). 50-/xmthick sections were serially collected in phosphate-buffered saline (PBS; pH 7.4) and stored free floating at 4°C until antibody incubations or Cresyl violet-staining. Prior to the immunocytochemical procedures, the sections were pretreated at room temperature with a 1% solution of hydrogen peroxide in PBS for 30 min and then rinsed 5 ×(10 min each) in PBS containing 0.2% Triton x-100 (PBS+T). Alternate sections were incubated overnight at 4°C in either rabbit anti-ChAT antisera diluted 1/1000 (AB 143; Chemicon Int) or anti-p75 LNGFR monoclonal antibody diluted 1/4000 (NGFr5, provided by M. Bothwell). The specificity of anti-ChAT AB 14315] and anti-p75 LN°FR33 antibodies has been previously described. The following incubations were performed at room temperature using PBS-T and rinses were 15 min each. The sections were washed twice after incubation with either of the two primary antibodies. For the ChAT immunocytochemistry, sections were then incubated for 1 h in biotinylated goat anti-rabbit serum (1/200; Vector Rabbit ABC kit), rinsed twice and incubated for 1 h in avidin-biotin complex (1/1000; Vector Rabbit ABC kit). For the p75 LN°FR immunocytochemistry, sections were incubated for 1 h in rabbit anti-mouse serum (1/50; prepared in our laboratory), given two rinses and incubated for 90 min in a solution containing monoclonal mouse antiperoxidase antibody (1/30; Medicorp, Canada) and 5/xg/ml horseradish peroxidase (HRP; Sigma, type IV). After two rinses, both sets of sections were incubated in a 0.06% solution of 3,3'-diaminobenzidine tetrahydrochloride (DAB; Sigma) for 15 min and then for an additional 5-10 min in the same solution containing 0.01% hydrogen peroxide. After three final rinses, sections were mounted onto chrome alum-subbed slides, dried, dehydrated through ascending concentrations of alcohol, cleared in xylene and coverslipped with Entellan (BDH, Que.). Throughout the length of the nucleus basalis, every third section was stained using Cresyl violet.

3

CHAT, nine p75 LNGFR- and nine Nissl-stained sections were independently analysed from each case (average 180 fields of 170× 220 /Lm/animal). In accordance with previous studies performed on rhesus monkeys [34,35], analysis of the C. aethiops nbM region (Ch4) was performed of its anteromedial (Ch4am), anterolateral (Ch4al), intermedioventral (Ch4iv), intermedio-dorsal (Ch4id) and posterior (Ch4p) subsectors. Sections of sham-operated and lesioned animals were carefully matched taking into account their anatomical characteristics. In Nissl-stained preparations, a frame shaped as the Ch4 subdivision of adjacent ChAT-immunostained sections was drawn over the nbM region to eliminate inappropriate sampling of neurons. The cross-sectional area and the number of CHAT- and p75LNGFR-immunoreactive neurons were calculated using an automatic edge-detection program [38]. The automatic counting was combined with manual editing which allows separation of contiguous cells, elimination of artifacts and vessel walls and lumens from the counting procedure. Only cell bodies with well-defined nucleus were included in the analysis [41]. The cell processes were measured by an automatic program able to discriminate neurites from the background. Detected neurites were skeletonized (i.e., reduced to single pixel lines) and the total length of neurites/unit area of tissue was computed per field. The total neurite length corresponded, therefore, to the entire network of processes present in the field [38]. In Nissl-stained preparations, all dyed magnocellular neurons with a diameter of > 20/zm and a clear neuronal-type nucleus were included for analysis, with the computer automatically excluding those that did not meet these criteria. The cell count was successively repeated in the same Ch4 subregions but without the arbitrary 20 /xm threshold. Statistical significance was tested between the two animal groups using ANOVA and a Newman-Keuls posthoc analysis.

2.4. Morphometry and statistics

3. Results The morphometrical analysis of CHAT-, p75 LNGFR- and Nisslstained sections was performed using a computerized image analysis system (Microcomputer Imaging Device - M1, Imaging Research, Brock University, Canada) connected to the stage of a Nikon microscope. Quantification, carried out at a total magnification of 25 × by two observers blind to the lesioned groups, involved the complete scan of all nbM subsectors in every third section [41]. A total of nine

Monkeys tolerated the surgery and the postoperative course was uncomplicated except for mild temporary right hemiparesis and hemianesthesia. They did not a p p e a r e d distressed and were able to feed themselves. The motor weakness was virtually undetectable

Table 1 Effect of cortical devascularization on cholinergic cell size and mean neurite length within different Ch4 subregions ipsilateral to lesioned side Group

Ch4am

ChAT-IR cell area (p,m 2) Sham 367 _+ 17 Lesioned 348 + 11 p75LNGFR-IR cell area (~m 2) Sham 356_+21 Lesioned 341 + 17 ChAT-IR neurite length a Sham 1811 _+89 Lesioned 1 720 _+65 p75LNOF~-IR neurite length a Sham 2134 -+ 78 Lesioned 1975 -+ 56

Ch4al

Ch4iv

Ch4id

Ch4p

373 + 16 304 _+ 21

381_+ 11 221_+ 19"

371_+ 15 232+_ 22"

369+11 335_+19

366+ 9 298 _+ 25

375+ 15 235-+ 14"

377+ 18 222-+ 17"

388-+ 9 348+23

1777+ 91 1563 + 123

1768+ 78 1124_+ 91"

1701+ 90 1089_+ 112"

1660+88 1497+97

2045 ± 75 1 895 -+ 65

2121+ 89 1387+112"

2003+ 1280+

1956+_94 1679+89

67 99"

The morphometrical analysis was carried out at a total magnification of 25 x , using a computerized image-analysis system (Microcomputer Imaging Device - M1). Values are expressed as mean + S.E.M in four sham-operated and in four cortical-devascularized animals. a Average'neurite length 5: S.E.M./field (170 x 220/zm). * P < 0.05 at ANOVA followed by a posthoc Newman-Keuls test.

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P. Liberini et al./Brain Research 648 (1994) 1-8

within 10-14 days after surgery. Although formal evaluation of sensory deficits was not performed, these seemed to persist longer. Six months following surgery, the animals were anesthetized and perfused as previously described. After removing the brains from the skull, a macroscopic study of the dissected hemispheres was carefully performed. In lesioned monkeys, a severely atrophied fronto-parieto-temporal area of the left hemisphere neocortex was detected. The infarction was limited to the superficial gyri. The cortex outside the lesioned area, the corpus callosum and other subcortical structures were preserved. In all cases, CHAT- and p75LNGFa-immunoreactive neurons were stained within the C. aethiops basal forebrain with the same distribution pattern and the nbM cytoarchitecture was analogous to that reported for other primates [34,35]. Thus, we adopted the classification system proposed by Mesulam et al. (1983) [34] to study the effects of the cortical lesion on each nbM subregion: anteromedial (Ch4am), anterolateral (Ch4al), intermedioventral (Ch4iv), intermedio-dorsal (Ch4id) and posterior (Ch4p). In sham-operated animals, the average cross-sectional area of ChAT and p 7 5 LNGFR n e u r o n s was not found to be significantly different in any of the Ch4 subregions (Table 1). In all lesioned monkeys significant damage of CHATand p75LNGFR-immunoreactive neurons occurred only in Ch4iv and Ch4id ipsilateral to the lesion (Table 1). Neuronal changes within these affected Ch4 regions predominantly consisted of cholinergic cell shrinkage and loss of neuritic processes. The cell bodies displayed a remarkable decrease of ChAT and p75 LNGFR immunoreactivity and the nuclei appeared eccentric, sometimes displacing the plasma membrane (Fig. 2). The computerized morphometrical analysis revealed that the average cross-sectional area of ChAT-immunoreactive neurons in Ch4iv and Ch4id ipsilateral to the lesion were significantly decreased to 58.0 + 5.0 and 62.5 + 5.9% of the sham-operated values (Table 1). Although not statistically significant, a trend toward decreased values of cross-sectional area also appeared in Ch4al (81.5 _+ 5.6% of the sham-operated values). The histogram in Fig. 3 shows that the majority of the cholinergic cells within the Ch4i of lesioned animals is smaller than those in corresponding areas of sham-operated animals. No significant changes of ChAT-immunoreactive cell size were detected in Ch4al, Ch4p and throughout the contralateral Ch4 subregions. Measurement of the cross-sectional area of p75LNGFR-im munoreactive neurons in the same regions displayed values of comparable magnitude (Table 1). The normally prominent CHAT- and p75LNGFR-im munoreactive neuropil observed in all the Ch4 regions of sham-operated animals was noticeably pale in Ch4iv and Ch4id of lesioned monkeys. Morphometric analysis

I

,/

L

Fig. 2. Effect of cortical lesion on morphology of p75LNGFR-immunoreactive neurons in Ch4iv region of C. aethiops: sham-operated (a) and cortical-devascularized monkey (b). Note cell shrinkage, eccentric displacement of nuclei, decrease of immunoreactivityand loss of neuritic processes in lesioned animals. Both micrographswere obtained with interference contrast optics. Bar = 10/~m.

confirmed this qualitative impression (Table 1). No significant changes of the fiber network were detected in any other Ch4 area of lesioned animals. Cholinergic cell count in Ch4 of sham-operated and lesioned monkeys was based on CHAT, p 7 5 LNGFR- and Nissl-stained sections. Quantitative analysis of sections stained with antibodies against CHAT- and p75 LNGFR did not demonstrate significant differences in cell numbers between lesioned and sham-operated animals in any Ch4 area (Table 2). In Nissl-stained sections of sham-operated animals, the Ch4 region was readily recognized as a prominent cluster of large, deeply stained ceils which extended from the anterior commissura, anteriorly, to the lateral geniculate nucleus, posteriorly. In lesioned brains, the number of magnocellular neurons within Ch4i appeared markedly reduced although this region was still easily recognizable for its great neuronal density. A significant reduction of Ch4iv and Ch4id cells (61.9 + 4.8 and 67.8 + 4.1%, respectively, of sham-operated

P. Liberini et al. / Brain Research 648 (1994) 1-8

4. D i s c u s s i o n

35-

30

S.am

~. 2o

~

15-

0

~

Cross SectionalArea (~m2) Fig. 3. Histogram showing cell size distribution of cholinergic cells within Ch4i of sham-operated (n = 4) and cortical-devascularized (n = 4) animals. Note that cortical lesion induced a shift in frequency towards a preponderance of small neurons.

values) was detected by the automated image analysis (see section 2) set for computing as magnocellular neurons cells with a perikaryal diameter of > 20 izm 2 (Table 2). In contrast, no significant reduction of Nissl-stained neurons was detected within the Ch4a, Ch4p and contralateral Ch4 subregions. The cell count was successively repeated using the same system but computing all cells displaying some Nissl material independently of their size. In this condition, no significant changes in cell number in any Ch4 region of lesioned monkeys were observed (Table 2).

Table 2 Effect of cortical devascularization on cholinergic cell density within different Ch4 subregions ipsilateral to lesioned side Group

5

Ch4am

ChAT-IR cells Sham 21.3+0.8 Lesioned 20.1+0.5 p75LNGFR-IR cells Sham 21.9_+0.4 Lesioned 21.5_+0.6 Nissl-stained cells a Sham 23.3-+0.8 Lesioned 21.4-+0.7 Nissl-stained cells b Sham 22.3_+0.9 Lesioned 23.3_+0.6

Ch4al

Ch4iv

Ch4id

Ch4p

22.5+0.6 21.7-+0.5 19.7+1.5 18.5+1.4

20.3+1.1 19.1+0.8

18.6+0.9 16.9_+0.8

22.3_+0.9 20.5:i:0.8 19.9_+0.8 18.8_+1.8

20.5_+1.2 19.3_+1.0 19.3_+1.1 17.8_+0.5

24.3_+0.8 23.1_+0.6 24.2+0.8 20.3:1:0.8 20.9-+ 1.7 14.3_+ 1.1 * 16.4_+ 1.0 * 18.9_+0.5 24.9_+0.8 24.8_+0.7 22.3+0.8 22.9_+0.8

24.7_+1.1 22.3_+1.9

21.5_+0.4 21.3_+0.8

Values are expressed as average number of cells _+S.E.M./field (170×220 /zm) in four sham-operated and in four cortical-devascularized animals. a Nissl-stained magnocellular neurons with a diameter of > 20/zm. b Nissl-stained neurons of any size. * P < 0.05 at ANOVA followed by a posthoc Newman-Keuls test.

The present study demonstrated that a focal infarction primarily affecting the C. aethiops neocortex leads to retrograde degeneration of neurons within the nucleus basalis complex. The morphometrical analysis revealed that only the intermediate Ch4 subregions and not other forebrain areas either ipsilateral or contralateral to the cortical lesion were affected as a result of the cortical infarction. This selective localization of retrograde degeneration is probably related to the topographical organization of the projections arising from the intermediate Ch4 region to the neocortex. As previously described [21,34,35], the Ch4id and Ch4iv subdivisions provide the major cholinergic input for the cortical areas corresponding to the surgically induced infarction. Thus, the disruption of terminal fields implicated in the synthesis and release of trophic factors might induce a retrograde degeneration mainly involving the intermediate Ch4 regions. Other potential mechanisms of neuronal injury, such as early intracellular disturbances of ion balance due to disruption of axonal membrane integrity, free radicals generation or release of excitatory amino acids, might operate in our lesion model [28]. During ischemia, the glutamate concentration at synapses surrounding the focal lesion can be increased for a sustained period of time, resulting in persistent stimulation of glutamate receptors that can induce secondary neuronal damage [31]. The morphological changes affecting the cholinergic neurons within the intermediate Ch4 region consisted of cell body shrinkage, eccentric displacement of the nuclei and loss of neuritic processes. Based on both ChAT and p75LN~FR-staining, no apparent loss of neurons in any of the Ch4 regions of lesioned monkeys was detected. In contrast, the automatic count, recognizing as magnocellular neurons Nissl-stained cells with a diameter of > 20/zm, revealed an apparent loss of cells within Ch4iv and Ch4id. The elimination of the arbitrary 20-/xm threshold allowed us to establish that no statistically significant loss of basophilic cells was present. This finding indicates that many shrunken but viable, Ch4i neurons were counted as lost using the perikaryal-size cell counting procedure. In contrast, by applying quantitative analysis to immunostained sections, both large and atrophied cholinergic neurons within the Ch4i region were counted and could, therefore, generate a more accurate reflection of the nbM's status. Accordingly, we would like to stress the reliability of nbM cell counts based on ChAT and p75 LNGFR immunoreactivity in experimental models of neurodegeneration as well as in human neuropathology. The lack of cell loss differentiates the present model from other experimental procedures, such as the fimbria fornix transection (for review, see Cuello et al. [9]). Recent investigations performed in primates using the

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P. Liberini et al./Brain Research 648 (1994) 1-8

latter experimental paradigm have shown that the surgical deafferentation of the hippocampus induces the disappearance of septal cholinergic neurons [24,53,54]. However, in this case, it is rather difficult to assess the actual balance between atrophy and cell death because the reported loss of ChAT and p75 LNGFR immunoreactivity might simply represent deficits in phenotypic expression of eholinergic markers a n d / o r neurotrophin receptors. The degenerative processes affecting the septal neurons are most likely the consequence of the experimental axotomy which induces a direct neuronal insult and a dramatic decrease of retrogradely transported trophic factors [15,59]. In contrast, in the cortical devascularizing lesion model, the morphological changes affecting the Ch4 region are related to disruption of the terminal target which consequently causes a gradual damage of the distal part of the axon [14]. Thus, the pathophysiological processes following the experimentally induced cortical infarction are more closely related to the nbM changes occurring in ischemic and degenerative brain disease than the transection of septo-hippocampal fibers. Interestingly, it has been reported that neurofibrillary tangle-bearing neurons were observed in the nbM of patients affected by cerebral infarction in the territory of the middle cerebral artery [19]. The finding that the presence of neurofibrillary tangles (NFT) was restricted to the nbM ipsilateral to the infarct suggested that in such a case the formation of NFT may occur as a retrograde reaction secondary to destruction of target tissue [19]. Recent in vivo studies indicate that the occurrence of retrograde neuronal death is largely dependent upon the distance of axonal injury from the cell body [46,52]. It is possible that the lack of neuronal death observed in our ischemic model is related to restriction of lesioning to the terminal segment of axons. This notion is reinforced by evidence that septal neurons do not die when their target is fully removed with the application of the excitotoxin amino acid N-methyl-D-aspartic acid but do degenerate after axotomy [47]. Alternatively, the widespread distribution of the nbM-to-cortex cholinergic projections in the monkey [21,35,36] could allow Ch4 neurons to continue receiving trophic support from cortical areas unaffected by the lesion. Furthermore, it is also possible that the survival of Ch4 neurons is assisted by synthesis of neurotrophic factors locally. Such a possibility is supported by the finding of increased NGF levels within the nbm region of cortically lesioned rats [30]. On the other hand, target-derived trophic factors have been postulated to be more important in maintaining phenotypic expression rather than cell viability [47]. It, therefore, seems that mature forebrain neurons are dependent on neurotrophic factors for the expression of cholinergic markers and for maintenance of shape, size and, presumably, synaptic connections [14]. However, it is possible that a survival

period longer than the 6 months used in our experimental design would disclose neuronal depletion. This issue deserves experimental attention in primates because shrunken neurons may be amenable to neurotrophic therapy in stages of the disease in which the molecular events leading to cell death are not yet activated. Further research should be aimed also at evaluating potential effects of the devascularizing lesion on non-cholinergic systems arising from subcortical nuclei to the neocortex. Such a comparative investigation might provide information on hierarchal vulnerability of specific neuronal populations to cortical target removal. In conclusion, the cortical devascularizing lesion is a potential model of infarction with which the cascade of retrograde degenerative processes within the nbM can be studied. Although further investigations are necessary to gain conclusive results, the present findings suggest that neocortical target cells may regulate the trophism of nbM cholinergic neurons but may not be essential for neuronal survival in the adult primate. The persistence of shrunken but viable nbM neurons might offer a possibility to asses potential reparative properties of an early as well as a delayed administration of neurotrophic agents.

Acknowledgements

This study was supported by funding from the Canadian Centres of Excellence Network for Neural Regeneration and Functional Recovery and the Medical Research Council (Canada). The monoclonal antibody anti-p75 LNGFR was kindly provided by M. Bothwell. We thank Dr. Frank Ervin for supplying the monkeys and for providing postoperative care. We are grateful to L. Garofalo, A. Ribeiro-da-Silva and H. Manev for helpful suggestions. We especially thank S. C6t6 for his expert technical assistance and we are also grateful to O. Mckprang and to M. Warmuth for editorial and secretarial assistance and D. Rolling for artistic expertise.

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