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Increase in glia-derived nerve growth factor following destruction of hippocampal neurons
76
Brain Research, 560 (1991) 76-83 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0(h%-8993/91/$03.50 ADONIS 000689939116974Z
BRES 16974
Increase in glia-derived nerve growth factor following destruction of hippocampal neurons Charles Bakhit 1, Mark Armanini 1, Gregory L. Bennett 2, Wai Lee T. Wong 2, Stanley E. H a n s e n 3 and R o b i n Taylor 3 Departments of 1Developmental Biology, 2Immunology Research and Assay Technology and 3Histology Laboratory, Genentech, Inc., South San Francisco, CA 94080 (U.S.A.) (Accepted 30 April 1991) Key words: Nerve growth factor; Choline acetyltransferase; Hippocampus; Lesion; Glia; Glial fibrillary acidic protein
It is currently believed that under normal conditions hippocampal neurons synthesize nerve growth factor (NGF) which may provide trophic support for cholinergic neurons projecting from the basal forebrain. The concept that glial cells are mobilized to increase the production of NGF following destruction of hippocampal neurons was examined. Excitotoxin-induced destruction of the dorsal hippocampal neurons resulted in a massive and prolonged increase in NGF-like immunoreactivity (LI). Immunostaining for NGF-LI and the glial marker, glial fibrillary acidic protein (GFAP), revealed that the source of increased NGF-LI production following the lesion were reactive astrocytes. Thus, glial cells assume the role of providing trophic support following loss of target neurons. INTRODUCTION A l t h o u g h it has b e e n established that N G F is a target-derived neurotrophic factor for sympathetic and neural-crest derived sensory neurons 13"22"32, its role in the central nervous system, in particular the well defined septo-hippocampal cholinergic pathway is unresolved. Accumulating evidence such as the specific r e t r o g r a d e transport of N G F from the h i p p o c a m p a l target to the cell bodies in the basal forebrain 28"3°, the partial loss of cholinergic cell bodies in the septum following intracerebroventricular (i.c.v.) administration of neutralizing antibodies to N G F 34, and the preservation of transected s e p t o - h i p p o c a m p a l cholinergic neurons following i.c.v. infusion of N G F 14"29'36, support a neurotrophic role for N G E F u r t h e r m o r e , it has been shown that the target h i p p o c a m p a l neurons are the primary source of N G E The hybridizing m R N A for N G F has been localized in neurons of the dentate gyrus and the p y r a m i d a l cell layer of the hippocampus 1'9'1°. H o w e v e r , the view that the s e p t o - h i p p o c a m p a l neurons are d e p e n d e n t on trophic support from the hippocampus is not consistent with the recent observation that extensive destruction of the target neurons in the hippocampus did not result in loss of septal cholinergic neurons 31. This finding suggests that a neurotrophic factor
may not be required for the survival of these cholinergic neurons. A possibility that might resolve the above inconsistency is the hypothesis that in response to injury, non-neuronal elements may play a critical role in the production of neurotrophic factors. Based on the current knowledge that h i p p o c a m p a l neurons are the primary source of N G F , we tested this hypothesis by destroying the neuronal source of N G F and examining the production of N G F by glial cells. We r e p o r t here that destruction of h i p p o c a m p a l neurons resulted in a massive and prolonged increase in N G F - L I . Further, glial cells were found to be a source of this increased N G F - L I production. MATERIALS AND METHODS Animals and surgery Male Sprague-Dawley rats weighing 250--350 g were anesthetized with pentobarbital (Nembutal, 50 mg/kg i.p.) and placed in a David Kopf small animal stereotaxic apparatus. A 26-gauge Hamilton cannula was inserted into the right hippocampus through a burr hole in the calvarium. The coordinates based on the atlas of Paxinos and Watson were: 4.0 P; 2.8 L; 3.5 V. Quinolinic acid (150 nmol/1 gl, pH 7.4) or sterile PBS (1 #1, pH 7.4) were infused at a rate of 0.5 #l/min. Two minutes following the infusion, the injection cannula was slowly removed, the calvarium hole was closed with bone wax and the scalp was apposed with wound clips. At various time points the animals were sacrificed and the brains removed. The dorsal hippocampus was dissected and frozen at -70 °C until assayed.
Correspondence: C. Bakhit, Beckman Research Institute of the City of Hope, Division of Neurosciences, 1450 E. Duarte Road, Duarte, CA 91010, U.S.A.
77 NG F measurements Preparation o f tissue samples. Tissue samples were prepared according to a modified procedure of Korsching and Thoenen 19 with the following modifications in the homogenizing buffer constituents: 0.5% bovine serum albumin (BSA) and 0.1% gelatin. The tissue samples were homogenized with a Brinkrnann Polytron homogenizer at 0 °C in 250 #1 of homogenizing buffer. The homogenates were centrifuged for 3 min at 60,000 rpm at 4 *C in a Beckman TL-100 ultracentrifuge. The supernatants were diluted 1:1 with 20 nM CAC12/0.2% Triton X-100. Recombinant human N G F standards. Recombinant human NGF (rhNGF) was generously provided by Dr. Louis E. Burton at Genentech, Inc. Protein content of a purified sample was determined by quantitative amino acid analysis and bioactivity using the PC12 culture system 12 using murine NGF as a standard. In the PC12 assay, rhNGF showed stimulatory activity with an EC50 of 39 pg/ml. Dilution of this sample in phosphate buffered saline (PBS) containing 5 g/l of BSA, 0.5 ml/l of Tween-20 and 0.1 ml/l of thimersoi (assay diluent) were then used as standards. NGF enzyme immunoassay. Nerve growth factor-like immunoreactivity (NGF-LI) was determined by a specific two-site ELISA. The NGF antibody was raised in rabbits against recombinant human NGF and affinity purified on a rhNGF affinity column to increase the specificity of the antibody and reduce or eliminate the possibility of non-specific cross-reactivity 1~. It is important to note that several growth factors including brain derived growth factor and neurotrophin-3 showed no significant cross-reactivity with this antibody. Further, to test for potential loss of NGF during the processing and assay procedure, known concentrations of rhNGF were added to hippocampal samples before homogenization. Subsequent assay of these samples showed complete recovery of the added concentration. Microtiter plate wells (Maxisorb, Nunc, Kamstrup, Denmark) were coated with 100/A of affinity purified rabbit antirhNGF at 10/zg/ml in coating buffer (0.05 M Na2CO 3, pH 9.6) for 18 h at 4 °C. Excess antibody was removed and the non-specific binding sites were blocked by the addition of 150 #1 per well of PBS containing 5 g/l of BSA and 0.1 ml/l of thimersol (blocking buffer), followed by incubation at room temperature for at least 1 h. After washing the wells with PBS containing 0.5 ml/1 of Tween-20 and 0.1 ml/I of thimersol (wash buffer), standards and samples diluted in assay diluent were added to the wells in 100/~1 volumes and incubated for 2 h at room temperature. The wells were washed again with wash buffer and 100 #i aliquots of horseradish peroxidase labeled rabbit anti-rhNGF were added to each well. After an incubation of 2 h at room temperature, the plates were washed as above and incubated for 20 min with 100/~1 of orthophenylene diamine (Sigma) at 2.2 mmol/1 in PBS, pH 7.2 with 0.012% (v/v) hydrogen peroxide. Formation of color was stopped with 100/A/well 2.25 tool/1 sulfuric acid and the absorbance was read at 490 nm minus 405 nm background with an automatic plate reader (Molecular Devices). The data were reduced using a four-parameter curve fitting program developed at Genentech, Inc. based on a algorithm for least-squares estimation of non-linear parameters 24. The lower detection limit of the assay was 50 pg/ml. To test for potential loss of NGF during the processing and assay procedure, known concentrations of rhNGF were added to hippocampal samples before homogenization. Subsequent assay of these samples showed complete recovery of the added concentration. To determine the antigenic determinant our antibody recognizes, dorsal hippocampal segments from 6 control rats and 6 quinolinic acid treated rats (150 nmol; 1 week post injection), were pooled separately. Assay of dilutions of the supernatants showed linear curves that ran parallel to the standard curve (data not shown), suggesting that the antibody recognized the same epitope. Further, the curve for the quinolinic acid treated animals was shifted to the right indicating higher concentrations of NGF. ChAT activity measurements ChAT activity was determined by the Fonnum procedure 8. Briefly, a 4/~1 aliquot of each homogenate prior to the centrifuga-
tion for the NGF ELISA, was added to an equal volume of 1% Triton X-100. To this 20 #1 of the reaction mixture containing the following was added: 0.2 mM [3I-I]acetyl-CoA, 300 mM NaCI, 50 mM sodium phosphate buffer (pH 7.4), 8 mM choline chloride, 20 mM EDTA (pH 7.4) and 0.1 mM physiostigmine in 20 mM EDTA. The labeled aeetylcholine was diluted with unlabeled compound. The mixture was incubated for 15 rain at 37 *C. The reaction was terminated by washing the reaction mixture tubes with 5 ml of icecold 10 mM sodium phosphate buffer (pH 7.4) into a scintillation vial containing 2 ml Kalignost/acetonitrile (0.5%, w/v) reagent. A toluene based scintillation fluid (0.5% PPO, 0.02% dimethyl POPOP, w/v) was added and the samples counted. lmmunohistochemical procedures One week after the injection of quinolinic acid (150 nmol), normal and treated rats were anesthetized and perfused through the descending aorta with 50 ml of PBS followed by 400 ml of 4% paraformaidehyde + 5% sucrose in PBS at 4 *C. The brains were dissected, postfixed in the same solution for 0.5 h, and incubated 12 h each at 4 °C in 10%, 20% and 30% sucrose in PBS. The brains were then frozen in dry ice and stored at -70 *(2 until used. The brains were sectioned in a cryostat at -20 °C. Ten-/zm-thiek sections were cut throughout the dorsal hippocampus and thaw mounted on polylysine coated slides. The mounted sections were placed in ice cold PBS. In some cases where GFAP staining was performed, brains were fixed in 10% formalin and paraffin embedded, cut at 8 #m thickness, and counterstained with hematoxylin. For immunoperoxidase labeling of NGF or GFAP, the Vector Vectastain Elite ABC kit was used. All incubations were done at 4 °C. First, endogenous peroxidase activity was quenched by incubating sections for 45 min in 0.3% hydrogen peroxide in methanol. Following three 10 min washes with PBS, endogenous biotin was blocked using the Vector Avidin/Biotin Blocking Kit. Sections were washed and non-specific binding sites were blocked by incubating the sections for 2 h in 1.5% normal goat serum in PBS. After washing, sections were incubated in a humidified chamber for 24 h with the affinity purified polyelonal antibody to rhNGF (0.5 #g/ml) or an affinity purified polyclonal GFAP antibody (Advanced Immunochemical Services, Inc.). Sections were then washed and incubated overnight with the secondary biotinylated antibody. After washing, sections were incubated for 3 h in the avidin/biotinylated horseradish peroxidase reagent, washed and stained in the diaminobenzidine tetrahydrochloride peroxidase substrate solution.
RESULTS To d e s t r o y h i p p o c a m p a l n e u r o n s w i t h o u t affecting aff e r e n t t e r m i n a l s , t h e e x c i t o t o x i c a m i n o acid, q u i n o l i n i c acid, was s t e r e o t a x i c a l l y i n j e c t e d u n i l a t e r a l l y i n t o the dorsal h i p p o c a m p u s . C o n s i s t e n t with p r e v i o u s r e p o r t s 29, i n t r a h i p p o c a m p a l injections of q u i n o l i n i c acid (150 n m o l in 1 #1) d e s t r o y e d intrinsic h i p p o c a m p a l n e u r o n s . H i s t o logical analysis o f sections t h r o u g h o u t t h e dorsal hippoca m p u s at 7 days r e v e a l e d d e g e n e r a t i o n o f m o s t o f the neurons. Loss of neurons included both region superior and i n f e r i o r a n d t h e p y r a m i d a l cells in C A 1 and C A 3 - 4 were completely eliminated. Additionally the granule cells w e r e n o l o n g e r visible (Fig. 1). H o w e v e r , despite r e m o v a l o f h i p p o c a m p a l n e u r o n s , q u i n o l i n i c acid p r o d u c e d a 2-fold i n c r e a s e in N G F - L I at 1 day a n d a 7 - 1 0 fold i n c r e a s e at 4 and 7 days f o l l o w i n g the i n j e c t i o n (Fig. 2). N G F - L I r e t u r n e d to 2 - 3 fold a b o v e c o n t r o l v a l u e s at 14, 28 and 120 days. To insure c o m p l e t e d e s t r u c t i o n o f
78
Fig. 1. Destruction of dorsal hippocampal neurons by quinolinic acid (150 nmol, 1 week) as revealed by hematoxylin and eosin staining in (a) normal and (b) quinolinic acid-treated rats.
79 TABLE I Effects of quinolinic acid, kainic acid and colchicine treatment on NGF-LI in the dorsal hippocampus 1 week later
Number of animals is 6. Treatment
PBS Quinolinic acid Kainic acid Colchicine
Dose (nmol)
N G F-L I (nglg wet weighO
150 x 2 1.4 15.0
2.3 -+ 0.3 31.8 - 2.8* 10.9 -+ 2.5* 8.9 -+ 1.2"
* Significantly different from control, P < 0.001, Student's t-test.
neurons in the dorsal hippocampus, two injections of quinolinic acid were made at coordinate levels 3.0 P, 1.8 L, 3.5 V and 5.0 P, 3.0 L, 3.5 V (Paxinos and Watson)• NGF-LI levels in the dorsal hippocampus with this lesion paradigm were 13-fold higher than the PBS controls 7 days following the injections (Table I). Further, injection of two other neurotoxins, kainic acid and colchicine produced similar increases in NGF-LI 7 days following the lesion (Table I), thus supporting the view that the increase in NGF-LI is in response to neuronal degeneration. To assess the integrity of cholinergic fibers and terminals in the lesioned area, choline acetyltransferase (CHAT) activity, which is a marker for cholinergic neurons 16, was also measured in supernatant aliquots from the same tissue samples. As is also shown in Fig. 2, there
1400 1200 4o
tOO0
NGF-LI
-e- ChAT ACtivity
800 -
were no significant changes in ChAT activity at any time point. In order to determine the source of NGF-LI following destruction of hippocampal neurons, immunohistochemical localization of NGF-LI in the hippocampus was performed. An affinity purified polyclonal antibody to recombinant human N G F was used for the immunolabeling. As shown in Fig. 3a, NGF-LI was found in neurons of the pyramidal and granule cell layers of the hippocampus in sections from normal rats. Staining of non-neuronal cells was not readily apparent• Exclusion of the primary or secondary antibody from the labeling procedure resulted in a complete loss of the immunostaining (data not shown). Furthermore, preabsorption of the primary antibody onto rhNGF bound to affinity gel prevented the immunostaining (Fig. 3b). Quinolinic acidinduced lesions of the dorsal hippocampus resulted in destruction of the hippocampal neurons and loss of their N G F immunostaining (Fig. 3c). However, intensely labeled, astrocyte-like cells were found in the lesion area (Fig. 3c). Similar controls were performed on hippocampu s from quinolinic acid-treated rats. The results showed complete elimination of the staining in all cases (data not shown). Staining for glial fibrillary acidic protein (GFAP), a glial marker, was not readily detectable in the normal hippocampus. However, in agreement with the characteristic astrocytic reaction after neuronal injury 4'25'33, destruction of neurons resulted in the appearance of hypertrophied astrocytes scattered around degenerating neurons throughout the dorsal hippocampus (Fig. 3d). The appearance of these cells was similar to that of the N G F immunoreactive cells following the lesion. Alternate labeling of serial sections for NGF-LI (Fig. 3e) and GFAP (Fig. 30 confirmed the above observations and revealed that not all astrocytes produce NGE
o c
600'
DISCUSSION
400 • 200 0
|
i
2~0
10
3'0
120
days
Fig. 2. Time course of the effects of quinolinic acid on NGF levels and ChAT activity in the dorsal hippocampus. Values are expressed as percent of the respective phosphate buffered saline (PBS) group at a particular time point. Values are means _+ S.E.M., n = 6. The average PBS control group for NGF was 2.30 _+ 0.37 ng/g wet weight. All quinolinic acid group values for NGF were significantly higher than control, *P < 0.05, **P < 0.001, Student's t-test. The average PBS control group for ChAT activity was 3218 -+ 134 nmol/ g/h. It should be noted that the tissue weight of the dorsal hippocampus of the quinolinic acid-treated group decreased to 79% of control at 120 days; however, ChAT activity, expressed as nmol/ g/h, remained unchanged.
Our finding of a significantly increased production of NGF-LI by glial cells following elimination of hippocampal neurons supports our working hypothesis. Although in vitro studies 21'26'a7 have shown that glial cells are capable of producing NGF, this has not been demonstrated in vivo, nor has the increased production of glia-derived N G F following neuronal lesion. The possibility that residual intrinsic hippocampal neurons could account for the increase in NGF-LI seems unlikely given the extent of the neuronal damage produced by quinolinic acid, the loss of NGF-LI staining by neurons of the pyramidal and granule cell layers, and the predominance of intensely stained glial cells in the lesioned area. Additionally, the correlation we observed between increased NGF-LI pro-
80
Fig. 3. Immunocytoehemical localization of N G F and G F A P in the dorsal hippocampus of normal and quinolinic acid-treated rats. a: staining of pyramidal and granule cell neurons with antibody to N G F in hippocampus of normal rat. b: loss of specific staining of tissue from normal animals after preabsorption of the antibody on a r h N G F affinity column, c: same staining as in a, howex,er, on tissue from an animal that (legend continued opposite)
81
received quinolinic acid treatment, d: staining of astrocytes with antibody to G F A P in hippocampus of an animal that received quinolinic acid treatment. Staining of serial sections (thickness = 10/~m) with antibody to NGF (¢) and GFAP (f). Arrows indicate same cells in both sections. The central structure is a blood vessel which also shows staining of N G E a and c, x250; a and c insets, x400; h ×100; d, e and f, x400.
82 duction in response to increasing neuronal destruction suggests an extraneuronal source of N G F - L I . The fact that C h A T activity did not change 120 days following a quinolinic acid-induced lesion of the dorsal hippocampus indicates the persistence of cholinergic afferents projecting from the medial septum to the hippocampus. This observation is in agreement with a recent report 31 demonstrating the preservation of cholinergic neurons in the medial septum following N-methyl-D-aspartate-induced lesioning of the hippocampus. Previous studies 11"18'35 which reported about a 50% increase in hippocampal N G F following fimbria-fornix lesions have attributed the increase to the destruction of basal forebrain afferents; these afferents normally reduce N G F levels by retrograde axonal transport. However, our lesion paradigm did not destroy afferent cholinergic axons and terminals in that there was no decrease in hippocampal C h A T activity. This is consistent with previous studies demonstrating that quinolinic acid destroys cell bodies but not afferent axons and terminals 17. Further, the magnitude of the increase in N G F - L I we observed was much larger than that reported in the timbria-fornix lesion studies. In addition, pyramidal and granule layer neurons, which produce N G F under normal conditions, were lost, and glial cells showed intense staining following the lesion. Thus the increase in hippocampal N G F - L I in our study was most likely due to increased synthesis of the protein by glial cells. Furthermore, the fact that C h A T activity was not altered, and specific binding sites for NGF, although slightly diminished, were still present 2, suggests that N G F continued to provide trophic support for the basal forebrain cholinergic neurons. It should be noted that in situ hybridization with complementary probes for N G F have reported diminution of NGF-hybridizing m R N A in hippocampal areas 7 days following destruction of neurons by kainic acid or colchicine 1. However, only one time point was investigated in that study. It is conceivable that the m R N A for N G F is transiently elevated at earlier times following the lesion. In a recent study Gall and Isackson 9 reported a
REFERENCES 1 Ayer-LeLievre, C., Olson, L., Ebendal, L., Seiger, A. and Persson, H., Expression of the beta-nerve growth factor gene in hippocampal neurons, Science, 240 (1988) 1339-1341. 2 Bakhit, C., Armanini, M., Dugich, M. and Altar, T., Receptor autoradiographic studies with 125I-rhNGF showed a 25% decrease in specific binding of labeled NGF 1 week following injection of 150 nmoles of quinolinic acid in the dorsal hippocampus, unpublished observations. 3 Bennett, G.L., Burton, L.E., Chan, W.P. and Wong, W.L.T., Sensitive enzyme immunoassay for recombinant human NGF, J. Cell. Biochem., Suppl. 14F (1990) 87.
transient increase in the m R N A for N G F in the granule cells of the hippocampus following limbic seizures produced by electrolytic lesion. The increase in the m R N A for N G F was maximal 2 - 6 h and declined at 24 h. Systematic investigations will be required to establish a correlation between regulation of the m R N A for N G F and expression of the protein. Our results demonstrate that glial cells play a critical role in the increased N G F - L I production, which continues to provide trophic support to cholinergic afferents following injury. Although target neurons may serve as the primary source of N G F under normal conditions, non-neuronal elements such as glia may assume that role following injury. A similar situation exists in the periphery. For example, target cells provide N G F to the peripheral nerve in rodents and levels of N G F and its m R N A are barely detectable in the nerve under normal conditions 15. However, transection of the peripheral nerve resuls in production of large quantities of N G F by Schwann cells in the distal portion LS. The increased levels of N G F following injury may be viewed as an important response of glial cells to minimize neuronal damage and promote regeneration following a lesion 23'27. It is conceivable that other neurotrophic factor(s) may also be released following injury of hippocampal neurons. Increases in 'neurotrophic activity' following CNS injury has been described s'6, and the existence of another neurotrophic factor in the hippocampus has been suggested 7 . It is interesting to note that survival of tissue transplants in the CNS is optimal 1 week following injury 5'6, a time point at which N G F - L I in our study were highest. These observations warrant investigating the possibility that sustained levels of pharmacological doses of N G F may be of benefit in limiting the initial damage following trauma and enhancing the regenerative process in the CNS.
Acknowledgements. We would like to thank Dr. Louis G. Burton and his laboratory for providing rhNGF, and Shamiram Feinglass for technical assistance.
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Brain Research, 560 (1991) 76-83 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0(h%-8993/91/$03.50 ADONIS 000689939116974Z
BRES 16974
Increase in glia-derived nerve growth factor following destruction of hippocampal neurons Charles Bakhit 1, Mark Armanini 1, Gregory L. Bennett 2, Wai Lee T. Wong 2, Stanley E. H a n s e n 3 and R o b i n Taylor 3 Departments of 1Developmental Biology, 2Immunology Research and Assay Technology and 3Histology Laboratory, Genentech, Inc., South San Francisco, CA 94080 (U.S.A.) (Accepted 30 April 1991) Key words: Nerve growth factor; Choline acetyltransferase; Hippocampus; Lesion; Glia; Glial fibrillary acidic protein
It is currently believed that under normal conditions hippocampal neurons synthesize nerve growth factor (NGF) which may provide trophic support for cholinergic neurons projecting from the basal forebrain. The concept that glial cells are mobilized to increase the production of NGF following destruction of hippocampal neurons was examined. Excitotoxin-induced destruction of the dorsal hippocampal neurons resulted in a massive and prolonged increase in NGF-like immunoreactivity (LI). Immunostaining for NGF-LI and the glial marker, glial fibrillary acidic protein (GFAP), revealed that the source of increased NGF-LI production following the lesion were reactive astrocytes. Thus, glial cells assume the role of providing trophic support following loss of target neurons. INTRODUCTION A l t h o u g h it has b e e n established that N G F is a target-derived neurotrophic factor for sympathetic and neural-crest derived sensory neurons 13"22"32, its role in the central nervous system, in particular the well defined septo-hippocampal cholinergic pathway is unresolved. Accumulating evidence such as the specific r e t r o g r a d e transport of N G F from the h i p p o c a m p a l target to the cell bodies in the basal forebrain 28"3°, the partial loss of cholinergic cell bodies in the septum following intracerebroventricular (i.c.v.) administration of neutralizing antibodies to N G F 34, and the preservation of transected s e p t o - h i p p o c a m p a l cholinergic neurons following i.c.v. infusion of N G F 14"29'36, support a neurotrophic role for N G E F u r t h e r m o r e , it has been shown that the target h i p p o c a m p a l neurons are the primary source of N G E The hybridizing m R N A for N G F has been localized in neurons of the dentate gyrus and the p y r a m i d a l cell layer of the hippocampus 1'9'1°. H o w e v e r , the view that the s e p t o - h i p p o c a m p a l neurons are d e p e n d e n t on trophic support from the hippocampus is not consistent with the recent observation that extensive destruction of the target neurons in the hippocampus did not result in loss of septal cholinergic neurons 31. This finding suggests that a neurotrophic factor
may not be required for the survival of these cholinergic neurons. A possibility that might resolve the above inconsistency is the hypothesis that in response to injury, non-neuronal elements may play a critical role in the production of neurotrophic factors. Based on the current knowledge that h i p p o c a m p a l neurons are the primary source of N G F , we tested this hypothesis by destroying the neuronal source of N G F and examining the production of N G F by glial cells. We r e p o r t here that destruction of h i p p o c a m p a l neurons resulted in a massive and prolonged increase in N G F - L I . Further, glial cells were found to be a source of this increased N G F - L I production. MATERIALS AND METHODS Animals and surgery Male Sprague-Dawley rats weighing 250--350 g were anesthetized with pentobarbital (Nembutal, 50 mg/kg i.p.) and placed in a David Kopf small animal stereotaxic apparatus. A 26-gauge Hamilton cannula was inserted into the right hippocampus through a burr hole in the calvarium. The coordinates based on the atlas of Paxinos and Watson were: 4.0 P; 2.8 L; 3.5 V. Quinolinic acid (150 nmol/1 gl, pH 7.4) or sterile PBS (1 #1, pH 7.4) were infused at a rate of 0.5 #l/min. Two minutes following the infusion, the injection cannula was slowly removed, the calvarium hole was closed with bone wax and the scalp was apposed with wound clips. At various time points the animals were sacrificed and the brains removed. The dorsal hippocampus was dissected and frozen at -70 °C until assayed.
Correspondence: C. Bakhit, Beckman Research Institute of the City of Hope, Division of Neurosciences, 1450 E. Duarte Road, Duarte, CA 91010, U.S.A.
77 NG F measurements Preparation o f tissue samples. Tissue samples were prepared according to a modified procedure of Korsching and Thoenen 19 with the following modifications in the homogenizing buffer constituents: 0.5% bovine serum albumin (BSA) and 0.1% gelatin. The tissue samples were homogenized with a Brinkrnann Polytron homogenizer at 0 °C in 250 #1 of homogenizing buffer. The homogenates were centrifuged for 3 min at 60,000 rpm at 4 *C in a Beckman TL-100 ultracentrifuge. The supernatants were diluted 1:1 with 20 nM CAC12/0.2% Triton X-100. Recombinant human N G F standards. Recombinant human NGF (rhNGF) was generously provided by Dr. Louis E. Burton at Genentech, Inc. Protein content of a purified sample was determined by quantitative amino acid analysis and bioactivity using the PC12 culture system 12 using murine NGF as a standard. In the PC12 assay, rhNGF showed stimulatory activity with an EC50 of 39 pg/ml. Dilution of this sample in phosphate buffered saline (PBS) containing 5 g/l of BSA, 0.5 ml/l of Tween-20 and 0.1 ml/l of thimersoi (assay diluent) were then used as standards. NGF enzyme immunoassay. Nerve growth factor-like immunoreactivity (NGF-LI) was determined by a specific two-site ELISA. The NGF antibody was raised in rabbits against recombinant human NGF and affinity purified on a rhNGF affinity column to increase the specificity of the antibody and reduce or eliminate the possibility of non-specific cross-reactivity 1~. It is important to note that several growth factors including brain derived growth factor and neurotrophin-3 showed no significant cross-reactivity with this antibody. Further, to test for potential loss of NGF during the processing and assay procedure, known concentrations of rhNGF were added to hippocampal samples before homogenization. Subsequent assay of these samples showed complete recovery of the added concentration. Microtiter plate wells (Maxisorb, Nunc, Kamstrup, Denmark) were coated with 100/A of affinity purified rabbit antirhNGF at 10/zg/ml in coating buffer (0.05 M Na2CO 3, pH 9.6) for 18 h at 4 °C. Excess antibody was removed and the non-specific binding sites were blocked by the addition of 150 #1 per well of PBS containing 5 g/l of BSA and 0.1 ml/l of thimersol (blocking buffer), followed by incubation at room temperature for at least 1 h. After washing the wells with PBS containing 0.5 ml/1 of Tween-20 and 0.1 ml/I of thimersol (wash buffer), standards and samples diluted in assay diluent were added to the wells in 100/~1 volumes and incubated for 2 h at room temperature. The wells were washed again with wash buffer and 100 #i aliquots of horseradish peroxidase labeled rabbit anti-rhNGF were added to each well. After an incubation of 2 h at room temperature, the plates were washed as above and incubated for 20 min with 100/~1 of orthophenylene diamine (Sigma) at 2.2 mmol/1 in PBS, pH 7.2 with 0.012% (v/v) hydrogen peroxide. Formation of color was stopped with 100/A/well 2.25 tool/1 sulfuric acid and the absorbance was read at 490 nm minus 405 nm background with an automatic plate reader (Molecular Devices). The data were reduced using a four-parameter curve fitting program developed at Genentech, Inc. based on a algorithm for least-squares estimation of non-linear parameters 24. The lower detection limit of the assay was 50 pg/ml. To test for potential loss of NGF during the processing and assay procedure, known concentrations of rhNGF were added to hippocampal samples before homogenization. Subsequent assay of these samples showed complete recovery of the added concentration. To determine the antigenic determinant our antibody recognizes, dorsal hippocampal segments from 6 control rats and 6 quinolinic acid treated rats (150 nmol; 1 week post injection), were pooled separately. Assay of dilutions of the supernatants showed linear curves that ran parallel to the standard curve (data not shown), suggesting that the antibody recognized the same epitope. Further, the curve for the quinolinic acid treated animals was shifted to the right indicating higher concentrations of NGF. ChAT activity measurements ChAT activity was determined by the Fonnum procedure 8. Briefly, a 4/~1 aliquot of each homogenate prior to the centrifuga-
tion for the NGF ELISA, was added to an equal volume of 1% Triton X-100. To this 20 #1 of the reaction mixture containing the following was added: 0.2 mM [3I-I]acetyl-CoA, 300 mM NaCI, 50 mM sodium phosphate buffer (pH 7.4), 8 mM choline chloride, 20 mM EDTA (pH 7.4) and 0.1 mM physiostigmine in 20 mM EDTA. The labeled aeetylcholine was diluted with unlabeled compound. The mixture was incubated for 15 rain at 37 *C. The reaction was terminated by washing the reaction mixture tubes with 5 ml of icecold 10 mM sodium phosphate buffer (pH 7.4) into a scintillation vial containing 2 ml Kalignost/acetonitrile (0.5%, w/v) reagent. A toluene based scintillation fluid (0.5% PPO, 0.02% dimethyl POPOP, w/v) was added and the samples counted. lmmunohistochemical procedures One week after the injection of quinolinic acid (150 nmol), normal and treated rats were anesthetized and perfused through the descending aorta with 50 ml of PBS followed by 400 ml of 4% paraformaidehyde + 5% sucrose in PBS at 4 *C. The brains were dissected, postfixed in the same solution for 0.5 h, and incubated 12 h each at 4 °C in 10%, 20% and 30% sucrose in PBS. The brains were then frozen in dry ice and stored at -70 *(2 until used. The brains were sectioned in a cryostat at -20 °C. Ten-/zm-thiek sections were cut throughout the dorsal hippocampus and thaw mounted on polylysine coated slides. The mounted sections were placed in ice cold PBS. In some cases where GFAP staining was performed, brains were fixed in 10% formalin and paraffin embedded, cut at 8 #m thickness, and counterstained with hematoxylin. For immunoperoxidase labeling of NGF or GFAP, the Vector Vectastain Elite ABC kit was used. All incubations were done at 4 °C. First, endogenous peroxidase activity was quenched by incubating sections for 45 min in 0.3% hydrogen peroxide in methanol. Following three 10 min washes with PBS, endogenous biotin was blocked using the Vector Avidin/Biotin Blocking Kit. Sections were washed and non-specific binding sites were blocked by incubating the sections for 2 h in 1.5% normal goat serum in PBS. After washing, sections were incubated in a humidified chamber for 24 h with the affinity purified polyelonal antibody to rhNGF (0.5 #g/ml) or an affinity purified polyclonal GFAP antibody (Advanced Immunochemical Services, Inc.). Sections were then washed and incubated overnight with the secondary biotinylated antibody. After washing, sections were incubated for 3 h in the avidin/biotinylated horseradish peroxidase reagent, washed and stained in the diaminobenzidine tetrahydrochloride peroxidase substrate solution.
RESULTS To d e s t r o y h i p p o c a m p a l n e u r o n s w i t h o u t affecting aff e r e n t t e r m i n a l s , t h e e x c i t o t o x i c a m i n o acid, q u i n o l i n i c acid, was s t e r e o t a x i c a l l y i n j e c t e d u n i l a t e r a l l y i n t o the dorsal h i p p o c a m p u s . C o n s i s t e n t with p r e v i o u s r e p o r t s 29, i n t r a h i p p o c a m p a l injections of q u i n o l i n i c acid (150 n m o l in 1 #1) d e s t r o y e d intrinsic h i p p o c a m p a l n e u r o n s . H i s t o logical analysis o f sections t h r o u g h o u t t h e dorsal hippoca m p u s at 7 days r e v e a l e d d e g e n e r a t i o n o f m o s t o f the neurons. Loss of neurons included both region superior and i n f e r i o r a n d t h e p y r a m i d a l cells in C A 1 and C A 3 - 4 were completely eliminated. Additionally the granule cells w e r e n o l o n g e r visible (Fig. 1). H o w e v e r , despite r e m o v a l o f h i p p o c a m p a l n e u r o n s , q u i n o l i n i c acid p r o d u c e d a 2-fold i n c r e a s e in N G F - L I at 1 day a n d a 7 - 1 0 fold i n c r e a s e at 4 and 7 days f o l l o w i n g the i n j e c t i o n (Fig. 2). N G F - L I r e t u r n e d to 2 - 3 fold a b o v e c o n t r o l v a l u e s at 14, 28 and 120 days. To insure c o m p l e t e d e s t r u c t i o n o f
78
Fig. 1. Destruction of dorsal hippocampal neurons by quinolinic acid (150 nmol, 1 week) as revealed by hematoxylin and eosin staining in (a) normal and (b) quinolinic acid-treated rats.
79 TABLE I Effects of quinolinic acid, kainic acid and colchicine treatment on NGF-LI in the dorsal hippocampus 1 week later
Number of animals is 6. Treatment
PBS Quinolinic acid Kainic acid Colchicine
Dose (nmol)
N G F-L I (nglg wet weighO
150 x 2 1.4 15.0
2.3 -+ 0.3 31.8 - 2.8* 10.9 -+ 2.5* 8.9 -+ 1.2"
* Significantly different from control, P < 0.001, Student's t-test.
neurons in the dorsal hippocampus, two injections of quinolinic acid were made at coordinate levels 3.0 P, 1.8 L, 3.5 V and 5.0 P, 3.0 L, 3.5 V (Paxinos and Watson)• NGF-LI levels in the dorsal hippocampus with this lesion paradigm were 13-fold higher than the PBS controls 7 days following the injections (Table I). Further, injection of two other neurotoxins, kainic acid and colchicine produced similar increases in NGF-LI 7 days following the lesion (Table I), thus supporting the view that the increase in NGF-LI is in response to neuronal degeneration. To assess the integrity of cholinergic fibers and terminals in the lesioned area, choline acetyltransferase (CHAT) activity, which is a marker for cholinergic neurons 16, was also measured in supernatant aliquots from the same tissue samples. As is also shown in Fig. 2, there
1400 1200 4o
tOO0
NGF-LI
-e- ChAT ACtivity
800 -
were no significant changes in ChAT activity at any time point. In order to determine the source of NGF-LI following destruction of hippocampal neurons, immunohistochemical localization of NGF-LI in the hippocampus was performed. An affinity purified polyclonal antibody to recombinant human N G F was used for the immunolabeling. As shown in Fig. 3a, NGF-LI was found in neurons of the pyramidal and granule cell layers of the hippocampus in sections from normal rats. Staining of non-neuronal cells was not readily apparent• Exclusion of the primary or secondary antibody from the labeling procedure resulted in a complete loss of the immunostaining (data not shown). Furthermore, preabsorption of the primary antibody onto rhNGF bound to affinity gel prevented the immunostaining (Fig. 3b). Quinolinic acidinduced lesions of the dorsal hippocampus resulted in destruction of the hippocampal neurons and loss of their N G F immunostaining (Fig. 3c). However, intensely labeled, astrocyte-like cells were found in the lesion area (Fig. 3c). Similar controls were performed on hippocampu s from quinolinic acid-treated rats. The results showed complete elimination of the staining in all cases (data not shown). Staining for glial fibrillary acidic protein (GFAP), a glial marker, was not readily detectable in the normal hippocampus. However, in agreement with the characteristic astrocytic reaction after neuronal injury 4'25'33, destruction of neurons resulted in the appearance of hypertrophied astrocytes scattered around degenerating neurons throughout the dorsal hippocampus (Fig. 3d). The appearance of these cells was similar to that of the N G F immunoreactive cells following the lesion. Alternate labeling of serial sections for NGF-LI (Fig. 3e) and GFAP (Fig. 30 confirmed the above observations and revealed that not all astrocytes produce NGE
o c
600'
DISCUSSION
400 • 200 0
|
i
2~0
10
3'0
120
days
Fig. 2. Time course of the effects of quinolinic acid on NGF levels and ChAT activity in the dorsal hippocampus. Values are expressed as percent of the respective phosphate buffered saline (PBS) group at a particular time point. Values are means _+ S.E.M., n = 6. The average PBS control group for NGF was 2.30 _+ 0.37 ng/g wet weight. All quinolinic acid group values for NGF were significantly higher than control, *P < 0.05, **P < 0.001, Student's t-test. The average PBS control group for ChAT activity was 3218 -+ 134 nmol/ g/h. It should be noted that the tissue weight of the dorsal hippocampus of the quinolinic acid-treated group decreased to 79% of control at 120 days; however, ChAT activity, expressed as nmol/ g/h, remained unchanged.
Our finding of a significantly increased production of NGF-LI by glial cells following elimination of hippocampal neurons supports our working hypothesis. Although in vitro studies 21'26'a7 have shown that glial cells are capable of producing NGF, this has not been demonstrated in vivo, nor has the increased production of glia-derived N G F following neuronal lesion. The possibility that residual intrinsic hippocampal neurons could account for the increase in NGF-LI seems unlikely given the extent of the neuronal damage produced by quinolinic acid, the loss of NGF-LI staining by neurons of the pyramidal and granule cell layers, and the predominance of intensely stained glial cells in the lesioned area. Additionally, the correlation we observed between increased NGF-LI pro-
80
Fig. 3. Immunocytoehemical localization of N G F and G F A P in the dorsal hippocampus of normal and quinolinic acid-treated rats. a: staining of pyramidal and granule cell neurons with antibody to N G F in hippocampus of normal rat. b: loss of specific staining of tissue from normal animals after preabsorption of the antibody on a r h N G F affinity column, c: same staining as in a, howex,er, on tissue from an animal that (legend continued opposite)
81
received quinolinic acid treatment, d: staining of astrocytes with antibody to G F A P in hippocampus of an animal that received quinolinic acid treatment. Staining of serial sections (thickness = 10/~m) with antibody to NGF (¢) and GFAP (f). Arrows indicate same cells in both sections. The central structure is a blood vessel which also shows staining of N G E a and c, x250; a and c insets, x400; h ×100; d, e and f, x400.
82 duction in response to increasing neuronal destruction suggests an extraneuronal source of N G F - L I . The fact that C h A T activity did not change 120 days following a quinolinic acid-induced lesion of the dorsal hippocampus indicates the persistence of cholinergic afferents projecting from the medial septum to the hippocampus. This observation is in agreement with a recent report 31 demonstrating the preservation of cholinergic neurons in the medial septum following N-methyl-D-aspartate-induced lesioning of the hippocampus. Previous studies 11"18'35 which reported about a 50% increase in hippocampal N G F following fimbria-fornix lesions have attributed the increase to the destruction of basal forebrain afferents; these afferents normally reduce N G F levels by retrograde axonal transport. However, our lesion paradigm did not destroy afferent cholinergic axons and terminals in that there was no decrease in hippocampal C h A T activity. This is consistent with previous studies demonstrating that quinolinic acid destroys cell bodies but not afferent axons and terminals 17. Further, the magnitude of the increase in N G F - L I we observed was much larger than that reported in the timbria-fornix lesion studies. In addition, pyramidal and granule layer neurons, which produce N G F under normal conditions, were lost, and glial cells showed intense staining following the lesion. Thus the increase in hippocampal N G F - L I in our study was most likely due to increased synthesis of the protein by glial cells. Furthermore, the fact that C h A T activity was not altered, and specific binding sites for NGF, although slightly diminished, were still present 2, suggests that N G F continued to provide trophic support for the basal forebrain cholinergic neurons. It should be noted that in situ hybridization with complementary probes for N G F have reported diminution of NGF-hybridizing m R N A in hippocampal areas 7 days following destruction of neurons by kainic acid or colchicine 1. However, only one time point was investigated in that study. It is conceivable that the m R N A for N G F is transiently elevated at earlier times following the lesion. In a recent study Gall and Isackson 9 reported a
REFERENCES 1 Ayer-LeLievre, C., Olson, L., Ebendal, L., Seiger, A. and Persson, H., Expression of the beta-nerve growth factor gene in hippocampal neurons, Science, 240 (1988) 1339-1341. 2 Bakhit, C., Armanini, M., Dugich, M. and Altar, T., Receptor autoradiographic studies with 125I-rhNGF showed a 25% decrease in specific binding of labeled NGF 1 week following injection of 150 nmoles of quinolinic acid in the dorsal hippocampus, unpublished observations. 3 Bennett, G.L., Burton, L.E., Chan, W.P. and Wong, W.L.T., Sensitive enzyme immunoassay for recombinant human NGF, J. Cell. Biochem., Suppl. 14F (1990) 87.
transient increase in the m R N A for N G F in the granule cells of the hippocampus following limbic seizures produced by electrolytic lesion. The increase in the m R N A for N G F was maximal 2 - 6 h and declined at 24 h. Systematic investigations will be required to establish a correlation between regulation of the m R N A for N G F and expression of the protein. Our results demonstrate that glial cells play a critical role in the increased N G F - L I production, which continues to provide trophic support to cholinergic afferents following injury. Although target neurons may serve as the primary source of N G F under normal conditions, non-neuronal elements such as glia may assume that role following injury. A similar situation exists in the periphery. For example, target cells provide N G F to the peripheral nerve in rodents and levels of N G F and its m R N A are barely detectable in the nerve under normal conditions 15. However, transection of the peripheral nerve resuls in production of large quantities of N G F by Schwann cells in the distal portion LS. The increased levels of N G F following injury may be viewed as an important response of glial cells to minimize neuronal damage and promote regeneration following a lesion 23'27. It is conceivable that other neurotrophic factor(s) may also be released following injury of hippocampal neurons. Increases in 'neurotrophic activity' following CNS injury has been described s'6, and the existence of another neurotrophic factor in the hippocampus has been suggested 7 . It is interesting to note that survival of tissue transplants in the CNS is optimal 1 week following injury 5'6, a time point at which N G F - L I in our study were highest. These observations warrant investigating the possibility that sustained levels of pharmacological doses of N G F may be of benefit in limiting the initial damage following trauma and enhancing the regenerative process in the CNS.
Acknowledgements. We would like to thank Dr. Louis G. Burton and his laboratory for providing rhNGF, and Shamiram Feinglass for technical assistance.
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