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(Stroke. 1996;27:1609-1615.)
© 1996 American Heart Association, Inc.
Articles |
TGF-ß1 Protects Hippocampal Neurons Against Degeneration Caused by Transient Global Ischemia
Dose-Response Relationship and Potential Neuroprotective Mechanisms
the Department of Pharmacology and Toxicology, Philipps-University, Marburg, Germany.
Correspondence to Jochen H.M. Prehn, PhD, Department of Pharmacology and Toxicology, FB 16, Philipps-University, Ketzerbach 63, 35032 Marburg, Germany. E-mail prehn@mailer.uni-marburg.de.
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Abstract |
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Background and Purpose Transforming growth factor-ß1 (TGF-ß1) has been shown to rescue cultured neurons from excitotoxic and hypoxic cell death and to reduce infarct size after focal cerebral ischemia in mice and rabbits. The present study investigated the effects of TGF-ß1 in a different pathophysiological setting and the delayed neuronal death of hippocampal pyramidal cells after transient global ischemia in rats, and evaluated the potential mechanisms of the neuroprotective activity of TGF-ß1.
Methods Transient forebrain ischemia was induced in male adult Wistar rats with bilateral occlusion of both common carotid arteries combined with systemic hypotension for 10 minutes. Seven days after ischemia, brains were perfusion-fixed and stained for histological evaluation. TGF-ß1 or vehicle was injected intracerebroventricularly (ICV; 0.5, 4, and 50 ng) or intrahippocampally (4 ng) 1 hour before ischemia. For in vitro studies, hippocampal neurons were derived from E17 rat embryos and cultured for 10 to 14 days. Cells were exposed to (1) S-nitrosocysteine (SNOC; 30 µmol/L) to induce nitric oxide–induced oxidative injury and (2) staurosporine (0.03 µmol/L) to induce apoptotic cell death.
Results Transient forebrain ischemia caused extensive degeneration of CA1 hippocampal pyramidal cells in vehicle-treated control animals. Ischemic injury was not significantly reduced after ICV administration of 0.5 ng TGF-ß1 (71±7% damaged neurons versus 84±3% in vehicle-treated controls; n=9 and 11, respectively; P=.07, Mann-Whitney U test). Administration of 4 ng TGF-ß1 reduced the percentage of damaged CA1 pyramidal cells from 71±10% in controls to 52±7% in TGF-ß1–treated animals (n=11 and 12, respectively; P=.04). TGF-ß1 (4 ng) also produced significant protection when injected directly into the hippocampal tissue. In contrast, ICV administration of 50 ng TGF-ß1 failed to show a protective effect in two separate sets of experiments. In vitro, a 24-hour pretreatment of the cultured hippocampal neurons with TGF-ß1 (0.1 to 10 ng/mL) significantly inhibited both nitric oxide and staurosporine neurotoxicity. Posttreatment with TGF-ß1 (10 ng/mL) also inhibited staurosporine neurotoxicity but actually potentiated nitric oxide–induced neuronal injury.
Conclusions We demonstrated that TGF-ß1 in a surprisingly low dose range has the capacity to reduce injury to CA1 hippocampal neurons caused by transient global ischemia in rats. This protective action could well be associated with the antioxidative and antiapoptotic effects of TGF-ß1 demonstrated in vitro.
Key Words: apoptosis • cytokines • neuroprotection • nitric oxide • rats
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Introduction |
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TGF-ß1 is a ubiquitous cytokine that exerts biological effects on a variety of cell types,1 including microglia, astrocytes, and neurons.2 3 4 5 TGF-ß1 is involved in many physiological and pathophysiological processes, such as cell growth, differentiation, inflammation, and tissue repair. It is secreted in an inactive, latent form and can be activated by acidification, alkalinization, heat, or proteases.1 TGF-ß1 is minimally expressed in the intact brain, but its expression increases strongly after trauma, excitotoxicity, and ischemia/hypoxia.6 7 8 9 10
It is not yet clear whether the presence of this cytokine has deleterious or protective effects for neurons in a given pathophysiological condition.11 12 13 14 15 16 Several authors have demonstrated that TGF-ß1 has the capacity to rescue cultured neurons from excitotoxic and hypoxic cell death,13 14 to reduce infarct size after focal cerebral ischemia in mice and rabbits,14 15 and to ameliorate neuronal injury caused by combined hypoxia/ischemia in rats.16 On the other hand, TGF-ß1 has been suggested to contribute to neurodegeneration observed in HIV encephalopathy and Alzheimer's disease.11 12 In the first part of the present study, we investigated the effects of a range of concentrations of TGF-ß1 on the selective neuronal death of CA1 pyramidal neurons after transient forebrain ischemia in rats.
Apart from excitotoxic processes,17 18 an increased generation of free oxygen radicals19 20 and the activation of an endogenous cell death program ("apoptosis")21 are believed to be critically involved in the development of the delayed neuronal death of CA1 pyramidal neurons after cerebral ischemia. In the second part of our study, we elucidated potential neuroprotective mechanisms of TGF-ß1. To this end, we investigated the capacity of TGF-ß1 to inhibit oxidative and apoptotic injuries in cultured rat hippocampal neurons.
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Materials and Methods |
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Surgical Procedure
Transient forebrain ischemia was induced in male Wistar rats (270 to 320 g) under normothermic conditions with two-vessel occlusion combined with systemic hypotension according to the method of Smith et al22 and others.23 After anesthesia with 3.5% halothane, the rats were connected to a Starling-type respirator that delivered 1% halothane in a nitrous oxide/oxygen mixture (70%/30%). The carotid arteries were isolated, and the tail artery and jugular vein were cannulated. To prevent blood coagulation, heparin (200 IU/kg) was injected intravenously. Before the induction of ischemia, halothane was discontinued and muscle paralysis was maintained with suxamethonium chloride (2x5 mg/kg IV). Body temperature was maintained at 37°C, PO2 at above 100 mm Hg, and PCO2 at 35 to 40 mm Hg. Ischemia was induced by clamping both common carotid arteries and by reducing the mean arterial blood pressure to 40 mm Hg with central venous exsanguination and an injection of trimethaphan camphor sulfonate (10 mg/kg IV). After 10 minutes, the carotid clamps were removed and the shed blood was reinfused. To minimize systemic acidosis, the rats received 50 mg/kg NaHCO3 IV. Levels of arterial pH, PCO2, PO2 (Corning 178 blood gas analyzer, Corning Medical), and plasma glucose concentration (Beckmann Glucose Analyzer II) were measured 20 and 10 minutes before, as well as 10 minutes after, ischemia. The animals were removed from the respirator as soon as they regained spontaneous respiration.
All animal procedures were performed in accordance with official governmental guidelines.
Histology
For histological evaluation, rats were anesthetized with chloral hydrate 7 days after ischemia and perfused transcardially through the ascending aorta with 0.9% saline followed by phosphate-buffered saline containing 4% formaldehyde. Brains were removed, embedded in paraffin, and sectioned. The sections were stained with a mixture of 1% celestine blue and 1% acid fuchsin. Both intact and damaged (ie, acidophilic) pyramidal cells in the CA1 subfield were counted. The cell counting was performed in a blinded fashion. Neuronal injury was expressed as the percentage of damaged neurons.
In vivo experiments were analyzed by Mann-Whitney U test.
Administration of Drugs
TGF-ß1 was injected as an ICV bolus into the left lateral cerebral ventricle or directly into the hippocampal tissue. Rats were anesthetized with 1% to 2% halothane in a 70:30 mixture of nitrous oxide/oxygen 1 day before ischemia and placed into a stereotactic apparatus. A hole was drilled into the skull at the stereotactically marked locations for the lateral ventricle (0.7 mm posterior to bregma, 1.5 mm lateral to midline, 5 mm ventral from cranium) or the hippocampus (3.8 mm posterior to bregma, 2 mm lateral to midline, 3.0 mm ventral from cranium), covered with foil and plasticine, and finally fixed with dental cement. One hour before ischemia, the plasticine and foil were removed, and TGF-ß1 was injected by means of a Hamilton syringe and a 31-gauge needle. TGF-ß1 was dissolved in 2 µL of the vehicle (saline containing 0.1% bovine serum albumin and 4 mmol/L HCl). Controls were given the vehicle in the same manner. After injection the skull was covered with dental cement.
Cell Cultures
Cultured rat pyramidal hippocampal neurons were derived from embryonic day 17 Holtzman rat embryos, which were isolated and plated onto poly-L-lysine–coated glass coverslips.24 Cells were maintained in a serum-free, N2.1-supplemented Dulbecco's modified Eagle medium above a layer of secondary astrocytes. Cells used were between 10 and 14 days of cultivation.
Induction of Neuronal Injury In Vitro
Oxidative injury was induced in the cultured rat hippocampal neurons by adding the NO-generating agent SNOC (10 to 1000 µmol/L) into the culture medium. SNOC has previously been shown to induce oxidative injury in rat cerebrocortical cultures via the formation of peroxynitrite.25 SNOC was freshly prepared immediately before the exposure. Cell viability was determined 24 hours afterward by trypan blue exclusion. For this purpose, cultures were washed with HEPES-buffered saline and stained with 0.4% trypan blue for a period of 5 minutes. A total of 200 to 300 neurons were counted per coverslip. Only dark-stained neurons were considered damaged. Cell viability was expressed as a percentage of the cell viability of untreated controls, which ranged from 80% to 93%. Cell counts were performed in a blinded fashion.
To produce apoptotic neuronal injury, staurosporine was added to the culture medium in concentrations of 0.01 to 1 µmol/L. Staurosporine is a nonselective protein kinase inhibitor and has been shown to induce apoptosis in all transformed and nontransformed cells tested thus far, including cultured cerebrocortical neurons.26 27 Twenty-four hours after the addition of staurosporine, the extent of neuronal degeneration was evaluated. Healthy neurons were identified morphologically as those having phase-bright round to oval cell bodies, smooth appearance, and intact dendrites. Injured neurons were characterized by rough, irregularly shaped cell bodies, nuclear condensation, and fragmented dendrites.
In vitro results were analyzed by ANOVA followed by Tukey's test.
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ICV Injection of TGF-ß1 Protects Against Delayed Neuronal Death of CA1 Pyramidal Neurons Caused by Transient Forebrain Ischemia
The physiological parameters of TGF-ß1–treated animals were essentially the same as in controls (Table
).
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ICV injection of 0.5 ng TGF-ß1 did not significantly reduce the extent of cell death in the CA1 subfield of the hippocampus. Neuronal damage of CA1 neurons was 71±7% in TGF-ß1–treated animals versus 84±3% in vehicle-treated controls (n=9 and 11, respectively; P=.07) (Fig 1A). In contrast, administration of 4 ng TGF-ß1 afforded significant protection of the pyramidal cells in the CA1 subfield. Neuronal damage was reduced from 71±10% in controls to 52±7% in TGF-ß1–treated animals (n=11 and 12, respectively; P=.04) (Fig 1B).
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Surprisingly, administration of 50 ng TGF-ß1 into the left lateral ventricle failed to further reduce the percentage of damaged neurons in the CA1 subfield. Neuronal viability of CA1 pyramidal cells was 69±8% in vehicle-treated controls versus 69±6% in TGF-ß1–treated animals (n=12 and 10, respectively; P=.29) (Fig 1C
). These results were confirmed in a second set of experiments using 9 TGF-ß1–treated animals (50 ng ICV) and 8 vehicle-treated animals (P=.145).
TGF-ß1 Protects CA1 Pyramidal Cells Against Ischemic Injury When Injected Directly Into the Hippocampal Tissue
We were next interested in determining whether TGF-ß1 also exerts neuroprotective activity when administered directly into the hippocampal tissue. Intrahippocampal injection of 4 ng TGF-ß1 clearly protected the hippocampal neurons against ischemic injury. Neuronal damage was reduced from 80±6% in vehicle-treated animals to 50±11% in TGF-ß1–treated animals (n=6 and 8, respectively; P=.04) (Fig 2).
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Pretreatment but Not Posttreatment With TGF-ß1 Protects Cultured Rat Hippocampal Neurons Against NO-Induced Oxidative Injury
Exposure of the cultured rat hippocampal neurons to the NO-generating agent SNOC (10 to 1000 µmol/L) produced a dose-dependent decline in neuronal viability (Fig 3A), indicated by positive trypan blue staining. A 24-hour pretreatment with TGF-ß1 (1 to 10 ng/mL) produced significant neuroprotection in cultures damaged with 30 µmol/L SNOC (Fig 3B). In contrast, a posttreatment with TGF-ß1 starting 5 minutes after the addition of SNOC (30 µmol/L) exerted no protective effects but actually potentiated SNOC neurotoxicity at concentrations of 1 and 10 ng/mL (Fig 3C).
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Pretreatment and Posttreatment With TGF-ß1 Protects Cultured Rat Hippocampal Neurons Against Staurosporine-Induced Apoptotic Injury
Exposure of the cultured rat hippocampal neurons to 0.01 to 1 µmol/L staurosporine induced morphological changes characteristic of apoptotic cell death in a dose-dependent manner (Fig 4A). Apoptotic injury was verified by morphological criteria including nuclear pyknosis, chromatin condensation, and cell shrinkage, as well as by positive nick end labeling of double-stranded DNA breaks using terminal deoxynucleotidyl transferase (data not shown; see also Reference 27). Cultures pretreated with TGF-ß1 (1 to 10 ng/mL) for 24 hours showed significantly fewer signs of apoptotic neuronal degeneration after exposure to 0.03 µmol/L staurosporine (Fig 4B). Posttreatment with TGF-ß1 (10 ng/mL) starting 5 minutes after the addition of staurosporine (0.03 µmol/L) also produced a significant neuroprotection, albeit to a lesser extent as in the case of the pretreatment (Fig 4C). Posttreatment with 0.1 or 1 ng/mL of TGF-ß1 showed no protective effects.
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Discussion |
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Blocking diverse glutamate receptors and calcium channels and thereby inhibiting intracellular Ca2+ overloading has been shown to afford significant protection against ischemic neuronal injury.28 However, a pathophysiologically high intracellular Ca2+ concentration may be only one factor in the cascade of events leading to neuronal degeneration. Neurotrophic factors, such as NGF and FGF-2, have also been shown to reduce ischemic neuronal injury caused by transient global cerebral ischemia,29 30 31 supporting the concept that ischemic neurodegeneration involves multiple processes.
In the present study, we have demonstrated that the cytokine TGF-ß1 affords significant protection against the delayed neuronal death of CA1 pyramidal cells caused by transient global ischemia (Figs 1 and 2). The conditions under which TGF-ß1 rescued hippocampal neurons after transient forebrain ischemia, however, differ significantly from those of other growth factors. FGF-2 prevented the delayed neuronal death of hippocampal CA1 pyramidal neurons after transient ischemia in gerbils when given in a dose of 240 ng/d for a period of 4 days.29 This treatment sums up to a total dose of approximately 14 µg FGF-2/kg body wt. NGF ameliorated neuronal degeneration after transient common carotid artery occlusion in gerbils,30 and the effective dose was approximately 150 µg NGF/kg body wt. In our experiments, approximately 15 ng TGF-ß1/kg body wt rescued the hippocampal neurons after global ischemia, ie, approximately 1000 to 10 000 times less than for the above-mentioned factors. TGF-ß1 therefore appears to be an extremely potent neuroprotective factor.
TGF-ß1 has previously been shown to reduce the infarct size in mice and rabbits subjected to focal cerebral ischemia.14 15 In both studies, TGF-ß1 was effective if administered 1 or several hours before the ischemic insult. In contrast to the present global ischemia study, however, doses of 1 and 4 µg/kg body wt were required to obtain a protective effect. Moreover, in the study of Gross and coworkers,15 TGF-ß1 was administered systemically, ie, by an infusion into the internal carotid artery. Different routes of administration and the different pathophysiology of the ischemic insult may explain the requirement for different doses of TGF-ß1 in global and focal cerebral ischemia.
Interestingly, we could not augment the neuroprotective effect of TGF-ß1 by increasing the dose of the cytokine. Similar effects of TGF-ß1 have been reported in other studies.15 16 The underlying mechanism for this U-shaped dose-response relationship remains elusive. It is conceivable that negative effects of TGF-ß1 occur at higher concentrations that could counteract the protective effect of TGF-ß1. In this context, it should be mentioned that TGF-ß1 is an extremely pleiotropic factor that acts on virtually every cell type.1 Our data indicate that posttreatment with TGF-ß1 is in fact able to potentiate NO-induced neuronal injury (Fig 3C). It has also been shown that TGF-ß1 protects against rapidly triggered NMDA receptor–dependent excitotoxic neuronal injury but potentiates slowly triggered excitotoxic injury in cultured rat hippocampal neurons.32 The above are examples of effects that could limit the neuroprotective activity of TGF-ß in vivo.
What are the potential protective mechanisms of TGF-ß1 against ischemic neurodegeneration? Inhibition of microglia activation has been suggested to contribute to the neuroprotective activity of TGF-ß1 in cerebral ischemia,2 16 but TGF-ß1 may also act on astrocytes and neurons.3 4 5 13 14 Overactivation of neuronal glutamate receptors is thought to play a key role in the pathophysiology of ischemic neuronal injury, and both NMDA- and non-NMDA antagonists have revealed neuroprotective effects in several ischemic models.28 It has previously been reported that TGF-ß1 has the capacity to rescue cultured central neurons from NMDA receptor–mediated excitotoxic injury.13 24 32 This protective effect of TGF-ß1 could be related to a stabilization of neuronal Ca2+ homeostasis.24
The mechanism of glutamate neurotoxicity may partly be explained by the generation of free radicals. Lafon-Cazal et al33 showed an NMDA-induced rise in superoxide radicals that was Ca2+ dependent and directly related to NMDA-induced neurotoxicity. Superoxide reacts with NO to peroxynitrite, which is neurotoxic.25 34 Constitutive NO synthase is maximally stimulated at calcium levels well below those reached in neurons during cerebral ischemia, and a blockade of this enzyme significantly reduced the infarct volume after an ischemic insult.35 Homozygous mutant mice lacking gene expression of neuronal NO synthase have also been shown to have significantly decreased neuronal injury after cerebral ischemia.36 The protective effect of a TGF-ß1 pretreatment against NO-mediated injury of cultured hippocampal neurons (Fig 3B) therefore demonstrates that this factor also modulates processes that occur downstream of glutamate receptor overactivation/Ca2+ overloading and suggests that these effects may also contribute to the neuroprotective activity of TGF-ß1.
There is increasing evidence that apoptotic mechanisms are involved in the development of delayed neuronal death after transient cerebral ischemia. It has been shown that inhibitors of RNA and protein synthesis are able to reduce the extent of neuronal damage in the CA1 region of the hippocampus.37 Moreover, DNA fragmentation and morphological changes that are characteristic of apoptosis were detected in the selective vulnerable areas of the brain after global ischemia.21 Therefore, the neuroprotective effect of TGF-ß1 against staurosporine-induced apoptotic cell death may also have relevance for the neuroprotective mechanisms of TGF-ß1 in vivo. Interestingly, TGF-ß1 at 10 ng/mL was also effective if administered shortly after the addition of staurosporine. The antiapoptotic effects of TGF-ß1 could be explained by an upregulation of the Bcl-2 protein expression as shown previously in cultured rat hippocampal neurons.24 Preliminary in vivo experiments using immunohistochemistry, however, failed to detect an increase in hippocampal Bcl-2 protein expression 48 hours after ICV injection of 5 or 50 ng TGF-ß1 in rats (P.H.-N., J.H.M.P., J.K., unpublished data).
In conclusion, our experiments demonstrate that TGF-ß1 is able to prevent the degeneration of hippocampal CA1 pyramidal cells caused by transient global ischemia in a surprisingly low dose range. The neuroprotective effects of this cytokine against NO- and staurosporine-induced cell death in vitro suggest that the antioxidative and antiapoptotic qualities of this factor could contribute to the protective effects of TGF-ß1 observed in vivo.
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Acknowledgments |
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These experiments were supported by grants from the Deutsche Forschungsgemeinschaft (Pr 338/2-1 and Forschergruppe "Neuroprotektion") (Drs Prehn and Krieglstein). We thank Professor Richard J. Miller (Department of Pharmacological and Physiological Sciences, The University of Chicago, Ill) for his support, and Sandra Engel and Volker Hartung for technical assistance.
Received December 27, 1995; revision received March 26, 1996; accepted May 13, 1996.
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13. Prehn JHM, Peruche B, Unsicker K, Krieglstein J. Isoform-specific effects of transforming growth factor-ß on degeneration of primary neuronal cultures induced by cytotoxic hypoxia or glutamate. J Neurochem. 1993;60:1665-1672.[Medline] [Order article via Infotrieve]
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Editorial Comment
Dose-Response Relationship and Potential Neuroprotective Mechanisms
Duke University Medical Center, Durham, NC
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Introduction |
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There seems little doubt that developing effective therapeutic measures to limit neuronal death would have an enormous impact on the treatment of ischemic stroke. Toward this goal, a number of recent studies have demonstrated a beneficial effect of growth factors/cytokines such as TGF-ß1, NGF, and FGF on neuronal survival after permanent or transient ischemic events in experimental animals. In this issue, Henrich-Noack and colleagues show that TGF-ß1 protects hippocampal neurons after transient ischemia. Interestingly, the protective effect of TGF-ß1 was noted with low-dose TGF-ß1 but completely disappeared when higher doses of TGF-ß1 were used. Complicating matters further, TGF-ß1 had a differential effect on neuronal survival in vitro depending on the stimulus for cell death.
Although studies such as these are encouraging, they also demonstrate that much has yet to be learned about the biology of growth factors and their receptors in the central nervous system (CNS) in both health and disease. Current evidence suggests that growth factors such as NGF, platelet-derived GF, and FGF play important roles in the CNS during development. However, the persistent expression of growth factor receptors in the adult CNS and the trophic effects they have on cultured neurons suggest important roles for these factors in the maintenance of the mature CNS.1R 2R 3R 4R 5R 6R 7R It is likely that a clearer understanding of the roles of these factors in the mature CNS and the signaling mechanisms whereby they exert these actions will lead to a better understanding of their protective effects after neuronal damage. A greater understanding of the molecular mechanisms of neuronal cell death after ischemic injury would likewise greatly enhance efforts to understand the protective effects of growth factors after ischemic injury. Understanding the function of growth factors in the CNS, as well as the mechanisms of neuronal cell death, should facilitate efforts to develop growth factor receptor agonists or antagonists to rescue damaged neurons after ischemic insult and conceivably to regenerate lost neuronal tissues.
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Selected Abbreviations and Acronyms |
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Male Wistar rats received an ICV injection of 4 ng TGF-ß1 or vehicle 1 hour before the induction of ischemia. Physiological parameters were measured 10 minutes before induction of ischemia. Values are mean±SD of 11 vehicle- and 12 TGF-ß1–treated animals.
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References |
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