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Early neuronal expression of tumor necrosis factor-α after experimental brain injury contributes to neurological impairment
Journal of Neuroimmunology 95 Ž1999. 115–125
Early neuronal expression of tumor necrosis factor-a after experimental brain injury contributes to neurological impairment Susan M. Knoblach
), 1
, Lei Fan 1, Alan I. Faden
Georgetown Institute for CognitiÕe and Computational Sciences, Rm. EP-04, Research Building, Georgetown UniÕersity Medical Center, 3970 ReserÕoir Road, NW, Washington, DC 20007-2197, USA Received 25 September 1998; revised 7 December 1998; accepted 7 December 1998
Abstract Tumor necrosis factor-alpha ŽTNFa . is a pleiotropic cytokine involved in inflammatory cascades associated with CNS injury. To examine the role of TNFa in the acute pathophysiology of traumatic brain injury ŽTBI., we studied its expression, localization and modulation in a clinically relevant rat model of non-penetrating head trauma. TNFa levels increased significantly in the injured cortex at 1 and 4, but not at 12, 24 or 72 h after severe lateral fluid-percussion trauma Ž2.6–2.7 atm.. TNFa was not elevated after mild injury. At 1 and 4 h after severe TBI, marked increases of TNFa were localized immunocytochemically to neurons of the injured cerebral cortex. A small population of astrocytes, ventricular cells and microvessels, also showed positive TNFa staining, but this expression was not injury-dependent. Macrophages that were present in a hemorrhagic zone along the external capsule, corpus callosum and alveus hippocampus at 4 h after TBI did not express TNFa. Intracerebroventricular administration of a selective TNFa antagonist—soluble TNFa receptor fusion protein ŽsTNFR:Fc. Ž37.5 mg. —at 15 min before and 1 h after TBI, improved performance in a series of standardized motor tasks after injury. In contrast, intravenous administration of sTNFR:Fc Ž0.2, 1 or 5 mgrkg. at 15 min after trauma did not improve motor outcome. Collectively, this evidence suggests that enhanced early neuronal expression of TNFa after TBI contributes to subsequent neurological dysfunction. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Tumor necrosis factor-a; Traumatic injury; Brain injury; Antagonist; Cerebral cortex; Behavioral dysfunction
1. Introduction Tumor necrosis factor-alpha ŽTNFa . is a multifunctional inflammatory cytokine associated with host-defense and homeostatic responses to injury or invasion. In the injured CNS, such responses may have both pathobiological and adaptive consequences. TNFa is elevated in the serum and cerebrospinal fluid of humans after traumatic brain injury ŽTBI. ŽGoodman et al., 1990; Ross et al., 1994.. TNFa mRNA and TNFa are also elevated after injury in clinically relevant models of brain trauma, such as closed head impact ŽShohami et al., 1994. and lateral
) Corresponding author. Tel.: q1-202-6872526; Fax: q1-2026870617; E-mail: [email protected] 1 These authors contributed equally to this manuscript.
fluid-percussion ŽTaupin et al., 1993; Yakovlev and Faden, 1995; Fan et al., 1996.. In these models, TNFa increases occur from 1 to 4 h after injury. Although the cellular sources of this early elevation have not been described, the rapid response suggests that TNFa may be synthesized by resident CNS cells, rather than by infiltrating vascular elements. Involvement of TNFa in the pathobiology of TBI is suggested by observations that agents which block TNFa synthesis, such as pentoxifylline, HU-211 and interleukin10, improved neuronal survival andror neurological recovery after traumatic injury ŽShohami et al., 1997; Knoblach and Faden, 1998.. These treatments, however, influence multiple secondary injury mechanisms, and are not TNFa specific. More compelling evidence comes from work showing that selective TNFa inhibition via monomeric, solubilized TNF receptors prevented neuronal degeneration and improved neurological recovery after experimental closed head injury ŽShohami et al., 1996.. In addition, this
0165-5728r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 5 7 2 8 Ž 9 8 . 0 0 2 7 3 - 2
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treatment attenuated edema and blood–brain barrier breakdown. In contrast, recent preliminary studies in knock-out mice generally appear to support an adaptive andror neuroprotective function of TNFa after TBI. TNF receptor knock-out mice developed larger lesions and more extensive breakdown of the blood–brain barrier relative to wild type controls 7 days after contusion injury ŽSullivan et al., 1997.. Similarly, TNFa knock-out mice developed larger lesions and scored lower on cognitive and motor tests than did wild type controls, when examined from 7 to 14 days after injury ŽScherbel et al., 1997.. Similar paradoxes are found in data regarding the role of TNFa in secondary injury mechanisms associated with TBI. TNFa is implicated in injury-induced inflammatory events; these include activation of glia, endothelial cells and blood elements Žmacrophages, neutrophils. ŽArvin et al., 1995, 1996., and enhanced expression of multiple down-stream inflammatory factors ŽBoehme et al., 1996; Sterner-Kock et al., 1996.. Thus, TNFa may contribute to reactive astrocytosis, leukocyte extravasation, vasoconstriction ŽMegyeri et al., 1992., coagulation ŽEstrada et al., 1995. and increased blood-barrier permeability ŽRosenberg et al., 1995.. In addition, it has been shown to damage neurons ŽTalley et al., 1995; Sipe et al., 1996., oligodendrocytes ŽHisahara et al., 1997. and endothelial cells Žde Vries et al., 1996; Duchini et al., 1996.. However, other studies show that TNFa is not only neuroprotective, it also activates components of specific cell survival pathways ŽCheng et al., 1994; Mattson et al., 1995.. In addition, it induces synthesis of neuronal growth factors ŽGadient et al., 1990. and promotes neuronal regeneration ŽSchwartz et al., 1991., both of which are probably critical to functional restoration. The resolution of these apparent controversies requires a more complete understanding of the injury-induced TNFa response. Therefore, the present studies utilized a clinically relevant rat model of lateral fluid-percussion injury to further clarify the role of TNFa in TBI. First, we quantified TNFa expression over time as a function of injury severity in the ipsilateral cortex, utilizing a specific TNFa enzyme-linked immunoadsorbent assay. We then elucidated the cellular elements that express TNFa after injury, using immunocytochemical techniques. Finally, because data that address whether TNFa actually influences functional recovery after TBI in clinically relevant models are limited, we examined the effect of selective TNFa inhibition on neurological outcome. To achieve this goal, we utilized a newly developed dimeric construct of recombinant human soluble p80 Žtype II. TNF receptors linked to the Fc portion of human IgG1 ŽsTNFR:Fc.. sTNFR:Fc neutralizes the effects of TNFa in vitro, and is efficacious in human and rodent studies of rheumatoid arthritis ŽMoreland et al., 1997., endotoxemia ŽMohler et al., 1993., and acute clinical syndrome associated with OKT3 antirejection therapy ŽWee et al., 1997..
2. Materials and methods 2.1. Lateral fluid-percussion injury Male Sprague–Dawley rats Ž400 " 25 g. were anesthetized with sodium pentobarbital Ž70 mgrkg, i.p.., intubated and ventilated on room air. A catheter was surgically inserted into the femoral artery for monitoring of blood gases and pressure. A midline scalp incision was made, the underlying temporal muscles reflected, and a craniotomy was performed over the left parietal cortex, 5 mm from the lambda suture and 4 mm from the sagittal suture. The intact dura was subjected to percussion insult as previously detailed and briefly summarized below ŽMcIntosh et al., 1989.. After injury, the arterial catheter was removed, incisions were closed, and surgical recovery was observed for 4 h, under temperature-controlled conditions. The lateral fluid-percussion model of traumatic head injury entails delivery of a pressurized pulse of sterile saline, defined in atmospheres Žatm., to the exposed parietal cortex. This produces a transient Žms. deformation of the underlying tissue, as well as a secondary injury to subcortical areas. The pressure of the pulse can be adjusted to produce variable degrees of injury. Mild Ž1.2 atm., moderate Ž2.0 atm. and severe Ž2.6–2.7 atm. levels of injury were used in the ELISA experiment. Severe injury was used in the immunocytochemical and neurological recovery studies. Of the three injury levels used, only mild injury causes no neurological deficits. Moderate injury produces deficits in all motor tests that resolve approximately 2 weeks after trauma. Severe injury is characterized by chronic neurological deficits and cortical degeneration that last well beyond the 2-week survival period ŽMcIntosh et al., 1989..
Fig. 1. Time-course of TNFa expression as measured by ELISA in cytosolic extracts of injured cortex after severe TBI Ž2.6–2.7 atm.. Histograms illustrate means"S.E.M. Ž ns 4–6 per group. in shams and at various hours after injury as indicated on the x-axis. ) p- 0.05 vs. sham using ANOVA followed by Dunnett’s test.
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Fig. 2. TNFa staining in the injured cortex 4 h after severe TBI Ž2.6–2.7 atm.. ŽA. Regional expression in the outer cortex under low magnification Ž25 = .. Some of the positively stained cells are marked by arrows. ŽB. The same region at higher magnification Ž100 = .. ŽC. The same cortical region from sham control Ž100 = .. ŽD, E, F. Staining in the inner cortex, next to the corpus callosum Žsome positively stained cells marked by arrows; corpus callosum marked by darts. at 1 ŽD. and 4 h ŽE. after TBI, or in sham control ŽF. ŽD, E, F; 50 = ..
Fig. 3. Representative photomicrographs Ž150 = . show TNFa staining that appeared to be associated with the plasma membrane in some cells as shown in ŽA., but more commonly was punctate and granular as seen in ŽB..
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Animals involved in the neurological recovery experiments were administered drugs either intracerebroventricularly Ži.c.v.. or intravenously Ži.v... Drugs were injected i.c.v. via a microsyringe ŽOD s 0.35 mm. inserted into the left lateral ventricle ŽAP s 0.8 mm; L s 1.4 mm, both from Bregma; H s 4.2 mm from skull ŽPaxinos and Watson, 1986... For i.v. infusions, drugs were injected via a catheter implanted acutely into the femoral vein. Sham controls Žsham-injury. were subjected to all surgical procedures but experienced no fluid-percussion insult. Vehicle controls were injured and treated with drug diluent.
O.C.T. embedding media and frozen in isopentane Žy708C.. Later, seven micron sections were sliced in a cryostat Žy158C., and thaw-mounted onto aminoalkylsilated slides. Sections were taken at y3.2 through y3.7 from Bregma ŽPaxinos and Watson, 1986., which includes the region of injury-induced lesion in the parieto-temporal cortex. Immunohistochemical procedures were performed as previously described by Tchelingerian et al. ŽTchelingerian et al., 1993., with minor modifications. Freshly cut sections were fixed for 5 min in acetone Žy308C., rinsed briefly in PBS, pH 7.4, blocked for 30 min in 10% goat
2.2. Enzyme-linked immunoadsorbent assay (ELISA) Rats were killed at 1, 4, 12, 24 or 72 h Ž n s 5 per group. after severe injury. Animals subjected to mild or moderate injury were killed at 1 or 72 h Ž n s 3 per group. after TBI. Sham controls were killed 1 or 24 h Ž n s 3 per group. after sham-injury. Brains were removed and quickly dissected on ice. A punch technique was used to isolate ; 100 mg of cortical tissue that contained the injury site, as well as tissue proximal to this region. This tissue is known to eventually deteriorate, resulting in a cavitation lesion ŽCortez et al., 1989.. Cytosolic extracts were prepared from fresh tissue using the method of Andrews and Faller ŽAndrews and Faller, 1991.. Tissue was homogenized Ž7 vrw ratio. on ice for 1 min in 10 mM HEPES– KOH, pH 7.9 buffer containing 1.5 mM MgCl 2 , 10 mM KCl, 0.5 mM DTT, 1.0 mM AEBSF, 10 mM leupeptin, 10 mM pepstatin and 0.1% Nonidet 40. Homogenates were centrifuged at 12,000 = g for 4 min at 48C. The resulting supernatant was removed and frozen Žy708C. as 110 ml aliquots. Later, TNFa levels were assessed in duplicate 50 ml aliquots, using a specific ELISA ŽGenzyme, Cambridge, MA., according to the manufacturer’s instructions. The primary antibody in this ELISA cross-reacts specifically with rat TNFa; mouse and rat TNFa generate superimposable standard curves in this assay, both with correlation coefficients of G 0.99. The accuracy of this ELISA has been quantified by recovery experiments, which indicate that the assay detects ; 90% of a recombinant rat TNFa standard. Protein levels were determined by the method of Bradford ŽBradford, 1976., using reagents from Bio-Rad ŽHercules, CA.. Spectrophometry was performed utilizing a Ceres 900 microplate reader ŽBiotek Instruments, Winooski, VT.. In cases where TNFa levels were below detectability, the data were recorded as 0 pgrmg protein. 2.3. Double-label fluorescent immunohistochemistry Rats were killed at 1 or 4 h Ž n s 5 per group. after severe injury or 4 h after sham-injury Ž n s 4.. The brains were removed, and the injured hemisphere was coated with
Fig. 4. The majority of TNFa positive cells in the injured cortical region were neurons. Cells stained with anti-TNFa only are shown in ŽA.. In ŽB. and ŽC., seperate multiple exposures ŽFITC vs. rhodamine. of the same field Ž150=. after double-label immunocytochemistry revealed that TNFa positive cells ŽB., also stained positively with anti-NeuN ŽC., a neuronal marker. Arrows denote cells that express both TNFa and NeuN.
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serumr0.01% Triton, and incubated with primary antibodies in 0.1% BSA for 16–18 h Ž48C. in a humidified chamber. Sections were then rinsed 3 times in PBS, incubated for 30 min with secondary antibodies, and rinsed again. Coverslips were mounted with PDE ŽJohnson and Nogueira Araujo, 1981., and the sections viewed with a Zeiss Axioplan-2 fluorescent microscope. Blocking solution and antibody dilutions were prepared using Dulbecco’s PBS, pH 7.4. Controls for immunocytochemical staining included deletion of either 18 or 28 antibody. Staining was
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initially performed with TNFa antibody alone, rather than as part of a double-labeling procedure, to ensure that results did not reflect bleed-through of the alternate fluorescence. Antibodies used in these experiments were as follows. Polyclonal rabbit anti-mouse Žrecombinant. TNFa , aIP-400 Ž1:50 or 1:150. from Genzyme ŽCambridge, MA. was raised against full-length TNFa. This antibody demonstrates cross-reactivity with rat TNFa ŽMerrill et al., 1993; Tchelingerian et al., 1993. and TNFa neutralizing activity in L929 cell cytotoxicity assays ŽSmith
Fig. 5. Double-label immunocytochemistry with anti-TNFa ŽA and D. and anti-GFAP ŽB and E. in the corpus callosum reveal TNFa expression occasionally in astrocytes Ždarts., and in association with microvessels Žarrows. at 4 h after severe Ž2.6–2.7 atm. TBI. Representative anti-TNFa staining is shown in ŽA. Ž50 = . and ŽD. Ž150 = ., with anti-GFAP staining in the respective identical fields ŽB, E.. No difference in the number of stained cells, or in staining intensity, was observed between injured tissue or sham controls. Representative anti-TNFa staining from the same region of sham controls is shown in ŽC. Ž50 = . and ŽF. Ž150 = ..
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et al., 1990. and in vivo models of inflammation ŽFranks et al., 1991; Teti et al., 1993.. Cell type specific antibodies included anti-NeuN Ž1:100. ŽChemicon, Temecula, CA., a neuronal marker ŽMullen et al., 1992.; anti-ED1 Ž1:500., which recognizes a cytoplasmic antigen unique to monocytes and macrophages ŽDamoiseaux et al., 1994.; and anti-GFAP Ž1:100. ŽBoerhinger Mannheim, Indianapolis, IN., which selectively stains glial fibrillary proteins in astrocytes ŽDebus et al., 1983.. Specific cell types were identified by morphological characteristics and positive marker staining. Affinity-purified fluorescein ŽFITC.-conjugated goat anti-rabbit IgG and Texas Red-conjugated goat anti-mouse IgG served as secondary antibodies ŽAccurate Chemicals, Westbury, NY.. 2.4. Drug administration Two independent studies examined the effect of sTNFR:Fc on mortality and neurological recovery during a 14-day period after TBI. In one study, 37.5 mg of sTNFR:Fc Žtotal volume of 10 ml., or equivolume vehicle Ž n s 13 per group., was injected i.c.v. 15 min before and 1 h after severe TBI. In the second study, either 0.2 Ž n s 6., 1 Ž n s 13. or 5 Ž n s 6. mgrkg sTNFR:Fc Žtotal volume of - 1 ml. was injected into the femoral vein 15 min after severe TBI. These doses were selected based on a positive effect of similar doses against LPS administration in the mouse ŽMohler et al., 1993.. Controls received either vehicle Ž n s 6. or 1 mgrkg IgG Ž n s 13., as a control for nonspecific protein effects. sTNFR:Fc was supplied by Immunex ŽSeattle, WA. in a 25 mM Tris, 150 mM NaCl buffer, pH 7.4 Žspecific activity 1.2 = 10 6 Urmg..
pressed as pgrmg protein" S.E.M. Composite and individual motor test neuroscores of animals that survived the 14-day scoring period were examined using Kruskal–Wallis nonparametric ANOVA, followed by Mann–Whitney U-tests Žcorrected for ties. for comparisons of treatment vs. control at each time examined. Significance was set at p - 0.05. Data were expressed as median and individual scores.
3. Results TNFa levels in cytosolic extracts prepared from injured cortex were significantly increased at 1 and 4, but not at 12 or 24 h after severe injury ŽFig. 1.. There was a trend toward a biphasic response, with a secondary increase in TNFa 72 h after severe TBI, but this did not reach statistical significance. TNFa levels also significantly increased at 1 h Ž59.11 " 11.25 pgrmg., but not at 72 h after moderate TBI Ž15.82 " 4.21 pgrmg.. Mild injury was not associated with elevated TNFa levels at any time
2.5. Neurological eÕaluation Post-traumatic neurological deficits were evaluated by an individual blind to treatment at 1, 7 and 14 days after TBI, based on performance in three motor tests as previously detailed ŽFaden et al., 1989.. Briefly, these tests include measures of forelimb flexion to a perceived fall, trunk and limb resistance to a lateral push and the ability to maintain a standing position on an inclined plane for at least 5 sec. Scores ranged from 0 Žmaximal deficits. to 5 Žnormal. for each limb on every test. In addition, the inclined plane includes an additional score for placement in a vertical position. By combining all scores, a composite neuroscore, ranging from 0 to 35, was determined. This scoring system reliably detects treatment effects 7 and 14 days after injury ŽFaden et al., 1989; Faden and Tzendzalian, 1992.. 2.6. Data analyses ELISA data were subjected to square root transformation and analyzed via ANOVA followed by Dunnett’s post-hoc test for multiple comparisons. Data were ex-
Fig. 6. TNFa was not present in macrophages Žarrows. seen within a hemorrhagic zone along the external capsule, corpus callosum and alveus hippocampus at 4 h after severe Ž2.6–2.7 atm. TBI. Representative photomicrographs Ž50=. are of the same field; anti-TNFa staining is shown in ŽA., and anti-ED-1 Ža macrophage marker. staining is shown in ŽB.. Cells lining the venticles also expressed TNFa ŽA, darts. in sham controls and at 1 or 4 h after injury.
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were rarely found in sham controls. When present, these cells were almost exclusively in vessels, or in association with meninges, and were not TNFa positive. Animals treated with i.c.v. injections of sTNFR:Fc showed significantly better neurological recovery than vehicle treated control rats at 7 and 14 days following TBI. The treatment effect was robust; rats that received sTNFR:Fc performed significantly better both as reflected by composite neuroscores ŽFig. 7., and also on every individual motor test score at both 7 and 14 days, with the exception of the 7 day inclined plane test, where p s 0.06 ŽFig. 8..
Fig. 7. Effect of intracerebroventricular administration of sTNFR:Fc on neurological recovery after severe Ž2.6–2.7 atm. TBI. sTNFR:Fc Ž37.5 mg in total volume of 10 ml TrisrNaCl buffer. or vehicle was injected 15 min before and 1 h after TBI. Histograms represent median composite neuroscores and each dot represents an individual animal score. Open box s vehicle control group, ns12 animals; filled box ssTNFR:Fc treated group, ns11 animals. )) p- 0.001 vs. controls using Mann– Whitney U-tests after Kruskal–Wallis nonparametric ANOVA.
examined Ž1 h s 22.49 " 8.29 pgrmg, 72 h s 16.68 " 3.81 pgrmg.. TNFa positive cells were identified immunocytochemically in the parietotemporal cortex of the injured hemisphere at 1 and 4 h after severe TBI. These cells were distributed throughout the region that deteriorates after injury Žy3.2 to y3.7 from Bregma. ŽFig. 2.. The staining intensity of these cells was markedly increased compared with that seen in sham controls processed identically. Most of the TNFa-positive cortical cells morphologically resembled neurons, and also stained positively with anti-NeuN antibody ŽFig. 3., a neuronal marker. Under high magnification, neuronal TNFa staining was characterized by a punctate, granular appearance andror beading on the cell surface ŽFig. 4., although the latter was less commonly observed. A few TNFa positive neurons were also present in extreme dorsal portions of the ipsilateral striatum, in the hilus of the ipsilateral dentate gyrus, and in regions CA1, and CA3 of the pyramidal cell layer of the ipsilateral hippocampus Ždata not shown.. Occasionally, TNFa positive microvessels were observed in sham controls and at 1 and 4 h after injury ŽFig. 5.. These were primarily detected in the corpus callosum and subependymal zone, but were also occasionally present in the inner and outer cortex. In addition, a small population of astrocytes, mainly in the corpus callosum, but also in cortex and hippocampus, were TNFa positive in sham controls and at 1 and 4 h after injury ŽFig. 5.. Macrophages were present in the inner cortex next to the corpus callosum, a frequent site of hemorrhage acutely associated with injury, at 1 and 4 h after TBI ŽFig. 6.. These cells did not stain positively for TNFa. Macrophages
Fig. 8. Effect of intracerebroventricular administration of sTNFR:Fc on neurological recovery as assessed by individual motor test scores. Data are from the same study described in Fig. 7. sTNFR:Fc Ž37.5 mg in total volume of 10 ml TrisrNaCl buffer. or vehicle was injected 15 min before and 1 h after TBI. Histograms represent median neuroscores and each dot represents an individual animal score. Open box s vehicle control group, ns12 animals; filled box ssTNFR:Fc treated group, ns11 animals. ) p- 0.05, )) p- 0.01, ))) p- 0.001 vs. controls using Mann– Whitney U-tests after Kruskal–Wallis nonparametric ANOVA.
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Fig. 9. Effect of intravenous administration of sTNFR:Fc on neurological recovery after TBI. Histograms represent median composite neuroscores for each group. Dots represent the performance of individual animals. sTNFR:Fc 1 mgrkg in TrisrNaCl buffer Žfilled box., ns13, or equigram IgG Žclear box., ns6, was injected 15 min after TBI. The neurological recovery of IgG controls was not significantly different from that of vehicle treated animals Ždata not shown.. There were no statistically significant differences in neuroscore between any group, at any time examined.
In contrast, intravenous injection of sTNFR:Fc Ž0.2, 1, or 5 mgrkg. did not improve neurological recovery at any time after TBI, compared with IgG or vehicle control animals ŽFig. 9.. No significant improvement was observed in any individual neurological test at 1, 7, or 14 days after TBI Ždata not shown.. Recovery was not significantly different between control groups treated with either 1 mgrkg IgG or vehicle. Neither i.c.v. nor i.v. administration of sTNFR:Fc altered mortality Ždata not shown. or physiological outcome measures Žtemperature, blood pressure, etc.., which remained within normal ranges.
4. Discussion These data show that TNFa expression is elevated early after TBI by levels of injury that cause neurological dysfunction. This enhanced TNFa synthesis primarily occurs in injured cortical neurons. In addition, selective inhibition of such injury-induced TNFa expression improves neurological recovery. Our results, showing substantial elevations in TNFa at 1 and 4 h, but not at 12, 24 or 72 h after injury, are consistent with similar changes observed previously in the same model ŽTaupin et al., 1993.. However, the present studies differ from this work in several respects. TNFa was previously quantified in whole tissue extracts via bioassay. In the present study, we utilized an ELISA to specifically measure expression of TNFa in cytosolic extracts. This resulted in lower overall levels of TNFa , but proportionally higher elevations in TNFa expression after injury Ži.e.. 10-fold vs. 25-fold.. These data suggest that much of the injury-associated elevations of TNFa
reflect changes in TNFa specifically, presumably through de novo synthesis. This interpretation is consistent both conceptually and temporally with data showing that TNFa mRNA expression increases from 30 min to approximately 4 h after lateral fluid-percussion injury ŽYakovlev and Faden, 1995; Fan et al., 1996.. Similar observations have been made in other models of CNS trauma, such as closed head injury ŽShohami et al., 1994. and spinal cord contusion ŽYakovlev and Faden, 1995; Semple-Rowland et al., 1997.. A possible biphasic TNFa response is suggested by the trend toward a significant elevation at 72 h. However, time course studies of TNFa mRNA found no delayed increases from 1 to 21 days after TBI ŽYakovlev and Faden, 1995; Fan et al., 1996.. In addition, others have not detected secondary elevations in TNFa from 24 to 72 h after injury ŽTaupin et al., 1993.. The present data suggest a positive association between neurological impairment and TNFa expression, since neither functional deficits nor TNFa elevations occur after mild TBI. These findings, together with the immunocytochemical data, suggest that enhanced neuronal expression of TNFa is coincident with neurological impairment, as injured neurons were the major producers of TNFa early after severe TBI. This expression was localized to tissue that received the primary traumatic insult, and later shows histological damage. Although TNFa was identified in some non-neuronal elements, such expression was not altered by injury. These results are in concurrence with those noting early neuronal expression of TNFa after a penetrating lesion ŽTchelingerian et al., 1993.. In contrast, TNFa expression was recently examined from 6 h to 16 days after a mild contusion injury that did not cause neurologic deficits ŽHolmin et al., 1997.: TNFa was expressed primarily by astrocytes, days Ž4–6. after the initial insult. Although that work differs somewhat from the present data in terms of model and time course, together these studies suggest that injury level, especially with reference to resultant neurological deficits, may determine which cell types express TNFa , as well as the timing of this response. Although the data suggest that neuronal TNFa may be present as either cytosolic or membrane-associated forms, our methods lack the resolution necessary to make such a conclusion. Recent evidence indicates that alternate forms of TNFa may mediate differential responses ŽAkassoglou et al., 1997. and preferentially activate specific receptor subtypes, with their respective signalling cascades ŽGrell et al., 1995; Yuan, 1997.. Macrophages visualized along the external capsule, corpus callosum and alveus hippocampus at 1 and 4 h after TBI may reflect hemorrhage that frequently occurs at this location in our model. The absence of TNFa staining in these cells, together with the observation that TNFa levels do not increase at 24 or 72 h after TBI, when macrophage infiltration occurs, suggests that these cells are not major sources of post-traumatic TNFa increases.
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Beneficial effects of monomeric, soluble, TNFR II receptors have been observed after i.v. administration following closed head injury, where they improved neurological recovery and prevented neuronal damage ŽShohami et al., 1996.. The current data represent important additional evidence, utilizing a different model and selective antagonist, that TNFa contributes to functional impairment after TBI. sTNFR:Fc was quite effective i.c.v., but, even over a range of doses, failed to improve outcome when given i.v. This finding may reflect differences between monomeric and dimeric soluble receptor constructs. Because TNFa primarily exists in trimeric form, the dimeric structure of sTNFR:Fc results in substantially greater affinity for TNFa than monomeric TNFR II receptors ŽPeppel et al., 1991; Schoenfeld et al., 1991.. sTNFR:Fc is also a more potent functional antagonist of TNFa activity ŽMohler et al., 1993.. However, the Fc backbone that helps confer these properties, along with the additional receptor moiety, greatly increases the size of this molecule. This may limit its ability to cross the blood–brain barrier, presumably even when compromised. Our data, in concurrence with those of others, indicates that the presence of TNFa in the early period after TBI is detrimental to functional outcome. Yet, explanations as to why this is so are not obvious, especially when viewed in light of growing evidence showing beneficial effects of TNFa after acute CNS injury in transgenic models ŽBruce et al., 1996; Scherbel et al., 1997; Sullivan et al., 1997., and also in mechanistic studies ŽMattson et al., 1995.. There are several possible explanations for these disparate findings. First, the early rise in TNFa that occurs after TBI may be detrimental, because it increases levels beyond that needed for cellular protection. Thus, TNFa produced by many individual neurons, as part of a local autocrine or paracrine protective response, may accumulate regionally and ultimately reach cytotoxic levels. The observation that TNFa is elevated in CSF after TBI ŽRoss et al., 1994. supports the notion that a significant overproduction of TNFa may exist. Neurons are relatively resistant to TNFa cytotoxicity ŽCheng et al., 1994; D’Souza et al., 1995.; however, actual local concentrations that occur after TBI, which are unknown, may be neurotoxic. Moreover, other cell-types, such as oligodendrocytes and endothelial cells, are differentially sensitive to the toxic effects of TNFa ŽD’Souza et al., 1995; de Vries et al., 1996; Duchini et al., 1996., and under conditions of overproduction, damage to these cells may indirectly lead to tissue loss and worsened outcome. Second, TNFa may subserve differential physiological roles over time after injury. TNFa knock-out mice performed better on neurological tests given in the first 48 h after TBI than did their respective controls, even though weeks later, this same group subsequently performed significantly worse ŽScherbel et al., 1997.. Thus, the actions of TNFa may be detrimental during the acute phase of injury, but beneficial in later phases of the secondary injury process. Third, cellular effects of TNFa are likely
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to be altered by other ongoing secondary injury events. For example, TNFa enhances the toxicity of excitatory amino acids ŽChao and Hu, 1994., which are also increased early after TBI ŽFaden et al., 1989.. Also, some cell survival strategies induced by TNFa depend on protein synthesis ŽCheng et al., 1994; Mattson et al., 1995; Wallach, 1997.. Because synthetic processes are inhibited in cells stressed or damaged by CNS injury ŽBodsch et al., 1985; Thilmann et al., 1987., it is possible that such protective schemes, potentially initiated by TNFa , cannot be completed. Consistent with this idea, most of the known protective effects of TNFa have been observed in pre-treatment paradigms ŽCheng et al., 1994; Nawashiro et al., 1997.. Lastly, the cellular actions of TNFa are complex and subject to divergent regulation. Thus, identical cell types may respond differently to TNFa , depending on such influencing factors as activated receptor subtype ŽSipe et al., 1996; Shen et al., 1997., receptor proportion ŽBotchkina et al., 1997., and cross-talk between other intracellular pathways ŽYuan, 1997.. The effect of TBI on many of these variables is, as yet, largely unknown. In summary, the present data show that early, neuronal production of TNFa contributes to neurological dysfunction after TBI. This suggests that although TNFa has many diverse effects, it primarily subserves a pathophysiological role in the secondary injury processes that occur soon after CNS trauma.
Acknowledgements All procedures involving live animals were approved by the Georgetown University Animal Care and Use Committee and conducted according to the principles set forth in the Guide for the Care and Use of Laboratory Animals, prepared by the Committee on the Care and Use of Laboratory Animals of the Institute of Laboratory Resources, National Research Council ŽDHEW Pub aNIH 85-232985.. We thank Randi Goodnight and Susan Wishner for technical assistance. These studies were supported by grant aR49CCR3-6634-07 from the Centers for Disease Control to A.I. Faden. S.M. Knoblach was supported in part by NIH 5T32HD07459.
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Early neuronal expression of tumor necrosis factor-a after experimental brain injury contributes to neurological impairment Susan M. Knoblach
), 1
, Lei Fan 1, Alan I. Faden
Georgetown Institute for CognitiÕe and Computational Sciences, Rm. EP-04, Research Building, Georgetown UniÕersity Medical Center, 3970 ReserÕoir Road, NW, Washington, DC 20007-2197, USA Received 25 September 1998; revised 7 December 1998; accepted 7 December 1998
Abstract Tumor necrosis factor-alpha ŽTNFa . is a pleiotropic cytokine involved in inflammatory cascades associated with CNS injury. To examine the role of TNFa in the acute pathophysiology of traumatic brain injury ŽTBI., we studied its expression, localization and modulation in a clinically relevant rat model of non-penetrating head trauma. TNFa levels increased significantly in the injured cortex at 1 and 4, but not at 12, 24 or 72 h after severe lateral fluid-percussion trauma Ž2.6–2.7 atm.. TNFa was not elevated after mild injury. At 1 and 4 h after severe TBI, marked increases of TNFa were localized immunocytochemically to neurons of the injured cerebral cortex. A small population of astrocytes, ventricular cells and microvessels, also showed positive TNFa staining, but this expression was not injury-dependent. Macrophages that were present in a hemorrhagic zone along the external capsule, corpus callosum and alveus hippocampus at 4 h after TBI did not express TNFa. Intracerebroventricular administration of a selective TNFa antagonist—soluble TNFa receptor fusion protein ŽsTNFR:Fc. Ž37.5 mg. —at 15 min before and 1 h after TBI, improved performance in a series of standardized motor tasks after injury. In contrast, intravenous administration of sTNFR:Fc Ž0.2, 1 or 5 mgrkg. at 15 min after trauma did not improve motor outcome. Collectively, this evidence suggests that enhanced early neuronal expression of TNFa after TBI contributes to subsequent neurological dysfunction. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Tumor necrosis factor-a; Traumatic injury; Brain injury; Antagonist; Cerebral cortex; Behavioral dysfunction
1. Introduction Tumor necrosis factor-alpha ŽTNFa . is a multifunctional inflammatory cytokine associated with host-defense and homeostatic responses to injury or invasion. In the injured CNS, such responses may have both pathobiological and adaptive consequences. TNFa is elevated in the serum and cerebrospinal fluid of humans after traumatic brain injury ŽTBI. ŽGoodman et al., 1990; Ross et al., 1994.. TNFa mRNA and TNFa are also elevated after injury in clinically relevant models of brain trauma, such as closed head impact ŽShohami et al., 1994. and lateral
) Corresponding author. Tel.: q1-202-6872526; Fax: q1-2026870617; E-mail: [email protected] 1 These authors contributed equally to this manuscript.
fluid-percussion ŽTaupin et al., 1993; Yakovlev and Faden, 1995; Fan et al., 1996.. In these models, TNFa increases occur from 1 to 4 h after injury. Although the cellular sources of this early elevation have not been described, the rapid response suggests that TNFa may be synthesized by resident CNS cells, rather than by infiltrating vascular elements. Involvement of TNFa in the pathobiology of TBI is suggested by observations that agents which block TNFa synthesis, such as pentoxifylline, HU-211 and interleukin10, improved neuronal survival andror neurological recovery after traumatic injury ŽShohami et al., 1997; Knoblach and Faden, 1998.. These treatments, however, influence multiple secondary injury mechanisms, and are not TNFa specific. More compelling evidence comes from work showing that selective TNFa inhibition via monomeric, solubilized TNF receptors prevented neuronal degeneration and improved neurological recovery after experimental closed head injury ŽShohami et al., 1996.. In addition, this
0165-5728r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 5 7 2 8 Ž 9 8 . 0 0 2 7 3 - 2
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treatment attenuated edema and blood–brain barrier breakdown. In contrast, recent preliminary studies in knock-out mice generally appear to support an adaptive andror neuroprotective function of TNFa after TBI. TNF receptor knock-out mice developed larger lesions and more extensive breakdown of the blood–brain barrier relative to wild type controls 7 days after contusion injury ŽSullivan et al., 1997.. Similarly, TNFa knock-out mice developed larger lesions and scored lower on cognitive and motor tests than did wild type controls, when examined from 7 to 14 days after injury ŽScherbel et al., 1997.. Similar paradoxes are found in data regarding the role of TNFa in secondary injury mechanisms associated with TBI. TNFa is implicated in injury-induced inflammatory events; these include activation of glia, endothelial cells and blood elements Žmacrophages, neutrophils. ŽArvin et al., 1995, 1996., and enhanced expression of multiple down-stream inflammatory factors ŽBoehme et al., 1996; Sterner-Kock et al., 1996.. Thus, TNFa may contribute to reactive astrocytosis, leukocyte extravasation, vasoconstriction ŽMegyeri et al., 1992., coagulation ŽEstrada et al., 1995. and increased blood-barrier permeability ŽRosenberg et al., 1995.. In addition, it has been shown to damage neurons ŽTalley et al., 1995; Sipe et al., 1996., oligodendrocytes ŽHisahara et al., 1997. and endothelial cells Žde Vries et al., 1996; Duchini et al., 1996.. However, other studies show that TNFa is not only neuroprotective, it also activates components of specific cell survival pathways ŽCheng et al., 1994; Mattson et al., 1995.. In addition, it induces synthesis of neuronal growth factors ŽGadient et al., 1990. and promotes neuronal regeneration ŽSchwartz et al., 1991., both of which are probably critical to functional restoration. The resolution of these apparent controversies requires a more complete understanding of the injury-induced TNFa response. Therefore, the present studies utilized a clinically relevant rat model of lateral fluid-percussion injury to further clarify the role of TNFa in TBI. First, we quantified TNFa expression over time as a function of injury severity in the ipsilateral cortex, utilizing a specific TNFa enzyme-linked immunoadsorbent assay. We then elucidated the cellular elements that express TNFa after injury, using immunocytochemical techniques. Finally, because data that address whether TNFa actually influences functional recovery after TBI in clinically relevant models are limited, we examined the effect of selective TNFa inhibition on neurological outcome. To achieve this goal, we utilized a newly developed dimeric construct of recombinant human soluble p80 Žtype II. TNF receptors linked to the Fc portion of human IgG1 ŽsTNFR:Fc.. sTNFR:Fc neutralizes the effects of TNFa in vitro, and is efficacious in human and rodent studies of rheumatoid arthritis ŽMoreland et al., 1997., endotoxemia ŽMohler et al., 1993., and acute clinical syndrome associated with OKT3 antirejection therapy ŽWee et al., 1997..
2. Materials and methods 2.1. Lateral fluid-percussion injury Male Sprague–Dawley rats Ž400 " 25 g. were anesthetized with sodium pentobarbital Ž70 mgrkg, i.p.., intubated and ventilated on room air. A catheter was surgically inserted into the femoral artery for monitoring of blood gases and pressure. A midline scalp incision was made, the underlying temporal muscles reflected, and a craniotomy was performed over the left parietal cortex, 5 mm from the lambda suture and 4 mm from the sagittal suture. The intact dura was subjected to percussion insult as previously detailed and briefly summarized below ŽMcIntosh et al., 1989.. After injury, the arterial catheter was removed, incisions were closed, and surgical recovery was observed for 4 h, under temperature-controlled conditions. The lateral fluid-percussion model of traumatic head injury entails delivery of a pressurized pulse of sterile saline, defined in atmospheres Žatm., to the exposed parietal cortex. This produces a transient Žms. deformation of the underlying tissue, as well as a secondary injury to subcortical areas. The pressure of the pulse can be adjusted to produce variable degrees of injury. Mild Ž1.2 atm., moderate Ž2.0 atm. and severe Ž2.6–2.7 atm. levels of injury were used in the ELISA experiment. Severe injury was used in the immunocytochemical and neurological recovery studies. Of the three injury levels used, only mild injury causes no neurological deficits. Moderate injury produces deficits in all motor tests that resolve approximately 2 weeks after trauma. Severe injury is characterized by chronic neurological deficits and cortical degeneration that last well beyond the 2-week survival period ŽMcIntosh et al., 1989..
Fig. 1. Time-course of TNFa expression as measured by ELISA in cytosolic extracts of injured cortex after severe TBI Ž2.6–2.7 atm.. Histograms illustrate means"S.E.M. Ž ns 4–6 per group. in shams and at various hours after injury as indicated on the x-axis. ) p- 0.05 vs. sham using ANOVA followed by Dunnett’s test.
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Fig. 2. TNFa staining in the injured cortex 4 h after severe TBI Ž2.6–2.7 atm.. ŽA. Regional expression in the outer cortex under low magnification Ž25 = .. Some of the positively stained cells are marked by arrows. ŽB. The same region at higher magnification Ž100 = .. ŽC. The same cortical region from sham control Ž100 = .. ŽD, E, F. Staining in the inner cortex, next to the corpus callosum Žsome positively stained cells marked by arrows; corpus callosum marked by darts. at 1 ŽD. and 4 h ŽE. after TBI, or in sham control ŽF. ŽD, E, F; 50 = ..
Fig. 3. Representative photomicrographs Ž150 = . show TNFa staining that appeared to be associated with the plasma membrane in some cells as shown in ŽA., but more commonly was punctate and granular as seen in ŽB..
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Animals involved in the neurological recovery experiments were administered drugs either intracerebroventricularly Ži.c.v.. or intravenously Ži.v... Drugs were injected i.c.v. via a microsyringe ŽOD s 0.35 mm. inserted into the left lateral ventricle ŽAP s 0.8 mm; L s 1.4 mm, both from Bregma; H s 4.2 mm from skull ŽPaxinos and Watson, 1986... For i.v. infusions, drugs were injected via a catheter implanted acutely into the femoral vein. Sham controls Žsham-injury. were subjected to all surgical procedures but experienced no fluid-percussion insult. Vehicle controls were injured and treated with drug diluent.
O.C.T. embedding media and frozen in isopentane Žy708C.. Later, seven micron sections were sliced in a cryostat Žy158C., and thaw-mounted onto aminoalkylsilated slides. Sections were taken at y3.2 through y3.7 from Bregma ŽPaxinos and Watson, 1986., which includes the region of injury-induced lesion in the parieto-temporal cortex. Immunohistochemical procedures were performed as previously described by Tchelingerian et al. ŽTchelingerian et al., 1993., with minor modifications. Freshly cut sections were fixed for 5 min in acetone Žy308C., rinsed briefly in PBS, pH 7.4, blocked for 30 min in 10% goat
2.2. Enzyme-linked immunoadsorbent assay (ELISA) Rats were killed at 1, 4, 12, 24 or 72 h Ž n s 5 per group. after severe injury. Animals subjected to mild or moderate injury were killed at 1 or 72 h Ž n s 3 per group. after TBI. Sham controls were killed 1 or 24 h Ž n s 3 per group. after sham-injury. Brains were removed and quickly dissected on ice. A punch technique was used to isolate ; 100 mg of cortical tissue that contained the injury site, as well as tissue proximal to this region. This tissue is known to eventually deteriorate, resulting in a cavitation lesion ŽCortez et al., 1989.. Cytosolic extracts were prepared from fresh tissue using the method of Andrews and Faller ŽAndrews and Faller, 1991.. Tissue was homogenized Ž7 vrw ratio. on ice for 1 min in 10 mM HEPES– KOH, pH 7.9 buffer containing 1.5 mM MgCl 2 , 10 mM KCl, 0.5 mM DTT, 1.0 mM AEBSF, 10 mM leupeptin, 10 mM pepstatin and 0.1% Nonidet 40. Homogenates were centrifuged at 12,000 = g for 4 min at 48C. The resulting supernatant was removed and frozen Žy708C. as 110 ml aliquots. Later, TNFa levels were assessed in duplicate 50 ml aliquots, using a specific ELISA ŽGenzyme, Cambridge, MA., according to the manufacturer’s instructions. The primary antibody in this ELISA cross-reacts specifically with rat TNFa; mouse and rat TNFa generate superimposable standard curves in this assay, both with correlation coefficients of G 0.99. The accuracy of this ELISA has been quantified by recovery experiments, which indicate that the assay detects ; 90% of a recombinant rat TNFa standard. Protein levels were determined by the method of Bradford ŽBradford, 1976., using reagents from Bio-Rad ŽHercules, CA.. Spectrophometry was performed utilizing a Ceres 900 microplate reader ŽBiotek Instruments, Winooski, VT.. In cases where TNFa levels were below detectability, the data were recorded as 0 pgrmg protein. 2.3. Double-label fluorescent immunohistochemistry Rats were killed at 1 or 4 h Ž n s 5 per group. after severe injury or 4 h after sham-injury Ž n s 4.. The brains were removed, and the injured hemisphere was coated with
Fig. 4. The majority of TNFa positive cells in the injured cortical region were neurons. Cells stained with anti-TNFa only are shown in ŽA.. In ŽB. and ŽC., seperate multiple exposures ŽFITC vs. rhodamine. of the same field Ž150=. after double-label immunocytochemistry revealed that TNFa positive cells ŽB., also stained positively with anti-NeuN ŽC., a neuronal marker. Arrows denote cells that express both TNFa and NeuN.
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serumr0.01% Triton, and incubated with primary antibodies in 0.1% BSA for 16–18 h Ž48C. in a humidified chamber. Sections were then rinsed 3 times in PBS, incubated for 30 min with secondary antibodies, and rinsed again. Coverslips were mounted with PDE ŽJohnson and Nogueira Araujo, 1981., and the sections viewed with a Zeiss Axioplan-2 fluorescent microscope. Blocking solution and antibody dilutions were prepared using Dulbecco’s PBS, pH 7.4. Controls for immunocytochemical staining included deletion of either 18 or 28 antibody. Staining was
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initially performed with TNFa antibody alone, rather than as part of a double-labeling procedure, to ensure that results did not reflect bleed-through of the alternate fluorescence. Antibodies used in these experiments were as follows. Polyclonal rabbit anti-mouse Žrecombinant. TNFa , aIP-400 Ž1:50 or 1:150. from Genzyme ŽCambridge, MA. was raised against full-length TNFa. This antibody demonstrates cross-reactivity with rat TNFa ŽMerrill et al., 1993; Tchelingerian et al., 1993. and TNFa neutralizing activity in L929 cell cytotoxicity assays ŽSmith
Fig. 5. Double-label immunocytochemistry with anti-TNFa ŽA and D. and anti-GFAP ŽB and E. in the corpus callosum reveal TNFa expression occasionally in astrocytes Ždarts., and in association with microvessels Žarrows. at 4 h after severe Ž2.6–2.7 atm. TBI. Representative anti-TNFa staining is shown in ŽA. Ž50 = . and ŽD. Ž150 = ., with anti-GFAP staining in the respective identical fields ŽB, E.. No difference in the number of stained cells, or in staining intensity, was observed between injured tissue or sham controls. Representative anti-TNFa staining from the same region of sham controls is shown in ŽC. Ž50 = . and ŽF. Ž150 = ..
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et al., 1990. and in vivo models of inflammation ŽFranks et al., 1991; Teti et al., 1993.. Cell type specific antibodies included anti-NeuN Ž1:100. ŽChemicon, Temecula, CA., a neuronal marker ŽMullen et al., 1992.; anti-ED1 Ž1:500., which recognizes a cytoplasmic antigen unique to monocytes and macrophages ŽDamoiseaux et al., 1994.; and anti-GFAP Ž1:100. ŽBoerhinger Mannheim, Indianapolis, IN., which selectively stains glial fibrillary proteins in astrocytes ŽDebus et al., 1983.. Specific cell types were identified by morphological characteristics and positive marker staining. Affinity-purified fluorescein ŽFITC.-conjugated goat anti-rabbit IgG and Texas Red-conjugated goat anti-mouse IgG served as secondary antibodies ŽAccurate Chemicals, Westbury, NY.. 2.4. Drug administration Two independent studies examined the effect of sTNFR:Fc on mortality and neurological recovery during a 14-day period after TBI. In one study, 37.5 mg of sTNFR:Fc Žtotal volume of 10 ml., or equivolume vehicle Ž n s 13 per group., was injected i.c.v. 15 min before and 1 h after severe TBI. In the second study, either 0.2 Ž n s 6., 1 Ž n s 13. or 5 Ž n s 6. mgrkg sTNFR:Fc Žtotal volume of - 1 ml. was injected into the femoral vein 15 min after severe TBI. These doses were selected based on a positive effect of similar doses against LPS administration in the mouse ŽMohler et al., 1993.. Controls received either vehicle Ž n s 6. or 1 mgrkg IgG Ž n s 13., as a control for nonspecific protein effects. sTNFR:Fc was supplied by Immunex ŽSeattle, WA. in a 25 mM Tris, 150 mM NaCl buffer, pH 7.4 Žspecific activity 1.2 = 10 6 Urmg..
pressed as pgrmg protein" S.E.M. Composite and individual motor test neuroscores of animals that survived the 14-day scoring period were examined using Kruskal–Wallis nonparametric ANOVA, followed by Mann–Whitney U-tests Žcorrected for ties. for comparisons of treatment vs. control at each time examined. Significance was set at p - 0.05. Data were expressed as median and individual scores.
3. Results TNFa levels in cytosolic extracts prepared from injured cortex were significantly increased at 1 and 4, but not at 12 or 24 h after severe injury ŽFig. 1.. There was a trend toward a biphasic response, with a secondary increase in TNFa 72 h after severe TBI, but this did not reach statistical significance. TNFa levels also significantly increased at 1 h Ž59.11 " 11.25 pgrmg., but not at 72 h after moderate TBI Ž15.82 " 4.21 pgrmg.. Mild injury was not associated with elevated TNFa levels at any time
2.5. Neurological eÕaluation Post-traumatic neurological deficits were evaluated by an individual blind to treatment at 1, 7 and 14 days after TBI, based on performance in three motor tests as previously detailed ŽFaden et al., 1989.. Briefly, these tests include measures of forelimb flexion to a perceived fall, trunk and limb resistance to a lateral push and the ability to maintain a standing position on an inclined plane for at least 5 sec. Scores ranged from 0 Žmaximal deficits. to 5 Žnormal. for each limb on every test. In addition, the inclined plane includes an additional score for placement in a vertical position. By combining all scores, a composite neuroscore, ranging from 0 to 35, was determined. This scoring system reliably detects treatment effects 7 and 14 days after injury ŽFaden et al., 1989; Faden and Tzendzalian, 1992.. 2.6. Data analyses ELISA data were subjected to square root transformation and analyzed via ANOVA followed by Dunnett’s post-hoc test for multiple comparisons. Data were ex-
Fig. 6. TNFa was not present in macrophages Žarrows. seen within a hemorrhagic zone along the external capsule, corpus callosum and alveus hippocampus at 4 h after severe Ž2.6–2.7 atm. TBI. Representative photomicrographs Ž50=. are of the same field; anti-TNFa staining is shown in ŽA., and anti-ED-1 Ža macrophage marker. staining is shown in ŽB.. Cells lining the venticles also expressed TNFa ŽA, darts. in sham controls and at 1 or 4 h after injury.
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were rarely found in sham controls. When present, these cells were almost exclusively in vessels, or in association with meninges, and were not TNFa positive. Animals treated with i.c.v. injections of sTNFR:Fc showed significantly better neurological recovery than vehicle treated control rats at 7 and 14 days following TBI. The treatment effect was robust; rats that received sTNFR:Fc performed significantly better both as reflected by composite neuroscores ŽFig. 7., and also on every individual motor test score at both 7 and 14 days, with the exception of the 7 day inclined plane test, where p s 0.06 ŽFig. 8..
Fig. 7. Effect of intracerebroventricular administration of sTNFR:Fc on neurological recovery after severe Ž2.6–2.7 atm. TBI. sTNFR:Fc Ž37.5 mg in total volume of 10 ml TrisrNaCl buffer. or vehicle was injected 15 min before and 1 h after TBI. Histograms represent median composite neuroscores and each dot represents an individual animal score. Open box s vehicle control group, ns12 animals; filled box ssTNFR:Fc treated group, ns11 animals. )) p- 0.001 vs. controls using Mann– Whitney U-tests after Kruskal–Wallis nonparametric ANOVA.
examined Ž1 h s 22.49 " 8.29 pgrmg, 72 h s 16.68 " 3.81 pgrmg.. TNFa positive cells were identified immunocytochemically in the parietotemporal cortex of the injured hemisphere at 1 and 4 h after severe TBI. These cells were distributed throughout the region that deteriorates after injury Žy3.2 to y3.7 from Bregma. ŽFig. 2.. The staining intensity of these cells was markedly increased compared with that seen in sham controls processed identically. Most of the TNFa-positive cortical cells morphologically resembled neurons, and also stained positively with anti-NeuN antibody ŽFig. 3., a neuronal marker. Under high magnification, neuronal TNFa staining was characterized by a punctate, granular appearance andror beading on the cell surface ŽFig. 4., although the latter was less commonly observed. A few TNFa positive neurons were also present in extreme dorsal portions of the ipsilateral striatum, in the hilus of the ipsilateral dentate gyrus, and in regions CA1, and CA3 of the pyramidal cell layer of the ipsilateral hippocampus Ždata not shown.. Occasionally, TNFa positive microvessels were observed in sham controls and at 1 and 4 h after injury ŽFig. 5.. These were primarily detected in the corpus callosum and subependymal zone, but were also occasionally present in the inner and outer cortex. In addition, a small population of astrocytes, mainly in the corpus callosum, but also in cortex and hippocampus, were TNFa positive in sham controls and at 1 and 4 h after injury ŽFig. 5.. Macrophages were present in the inner cortex next to the corpus callosum, a frequent site of hemorrhage acutely associated with injury, at 1 and 4 h after TBI ŽFig. 6.. These cells did not stain positively for TNFa. Macrophages
Fig. 8. Effect of intracerebroventricular administration of sTNFR:Fc on neurological recovery as assessed by individual motor test scores. Data are from the same study described in Fig. 7. sTNFR:Fc Ž37.5 mg in total volume of 10 ml TrisrNaCl buffer. or vehicle was injected 15 min before and 1 h after TBI. Histograms represent median neuroscores and each dot represents an individual animal score. Open box s vehicle control group, ns12 animals; filled box ssTNFR:Fc treated group, ns11 animals. ) p- 0.05, )) p- 0.01, ))) p- 0.001 vs. controls using Mann– Whitney U-tests after Kruskal–Wallis nonparametric ANOVA.
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Fig. 9. Effect of intravenous administration of sTNFR:Fc on neurological recovery after TBI. Histograms represent median composite neuroscores for each group. Dots represent the performance of individual animals. sTNFR:Fc 1 mgrkg in TrisrNaCl buffer Žfilled box., ns13, or equigram IgG Žclear box., ns6, was injected 15 min after TBI. The neurological recovery of IgG controls was not significantly different from that of vehicle treated animals Ždata not shown.. There were no statistically significant differences in neuroscore between any group, at any time examined.
In contrast, intravenous injection of sTNFR:Fc Ž0.2, 1, or 5 mgrkg. did not improve neurological recovery at any time after TBI, compared with IgG or vehicle control animals ŽFig. 9.. No significant improvement was observed in any individual neurological test at 1, 7, or 14 days after TBI Ždata not shown.. Recovery was not significantly different between control groups treated with either 1 mgrkg IgG or vehicle. Neither i.c.v. nor i.v. administration of sTNFR:Fc altered mortality Ždata not shown. or physiological outcome measures Žtemperature, blood pressure, etc.., which remained within normal ranges.
4. Discussion These data show that TNFa expression is elevated early after TBI by levels of injury that cause neurological dysfunction. This enhanced TNFa synthesis primarily occurs in injured cortical neurons. In addition, selective inhibition of such injury-induced TNFa expression improves neurological recovery. Our results, showing substantial elevations in TNFa at 1 and 4 h, but not at 12, 24 or 72 h after injury, are consistent with similar changes observed previously in the same model ŽTaupin et al., 1993.. However, the present studies differ from this work in several respects. TNFa was previously quantified in whole tissue extracts via bioassay. In the present study, we utilized an ELISA to specifically measure expression of TNFa in cytosolic extracts. This resulted in lower overall levels of TNFa , but proportionally higher elevations in TNFa expression after injury Ži.e.. 10-fold vs. 25-fold.. These data suggest that much of the injury-associated elevations of TNFa
reflect changes in TNFa specifically, presumably through de novo synthesis. This interpretation is consistent both conceptually and temporally with data showing that TNFa mRNA expression increases from 30 min to approximately 4 h after lateral fluid-percussion injury ŽYakovlev and Faden, 1995; Fan et al., 1996.. Similar observations have been made in other models of CNS trauma, such as closed head injury ŽShohami et al., 1994. and spinal cord contusion ŽYakovlev and Faden, 1995; Semple-Rowland et al., 1997.. A possible biphasic TNFa response is suggested by the trend toward a significant elevation at 72 h. However, time course studies of TNFa mRNA found no delayed increases from 1 to 21 days after TBI ŽYakovlev and Faden, 1995; Fan et al., 1996.. In addition, others have not detected secondary elevations in TNFa from 24 to 72 h after injury ŽTaupin et al., 1993.. The present data suggest a positive association between neurological impairment and TNFa expression, since neither functional deficits nor TNFa elevations occur after mild TBI. These findings, together with the immunocytochemical data, suggest that enhanced neuronal expression of TNFa is coincident with neurological impairment, as injured neurons were the major producers of TNFa early after severe TBI. This expression was localized to tissue that received the primary traumatic insult, and later shows histological damage. Although TNFa was identified in some non-neuronal elements, such expression was not altered by injury. These results are in concurrence with those noting early neuronal expression of TNFa after a penetrating lesion ŽTchelingerian et al., 1993.. In contrast, TNFa expression was recently examined from 6 h to 16 days after a mild contusion injury that did not cause neurologic deficits ŽHolmin et al., 1997.: TNFa was expressed primarily by astrocytes, days Ž4–6. after the initial insult. Although that work differs somewhat from the present data in terms of model and time course, together these studies suggest that injury level, especially with reference to resultant neurological deficits, may determine which cell types express TNFa , as well as the timing of this response. Although the data suggest that neuronal TNFa may be present as either cytosolic or membrane-associated forms, our methods lack the resolution necessary to make such a conclusion. Recent evidence indicates that alternate forms of TNFa may mediate differential responses ŽAkassoglou et al., 1997. and preferentially activate specific receptor subtypes, with their respective signalling cascades ŽGrell et al., 1995; Yuan, 1997.. Macrophages visualized along the external capsule, corpus callosum and alveus hippocampus at 1 and 4 h after TBI may reflect hemorrhage that frequently occurs at this location in our model. The absence of TNFa staining in these cells, together with the observation that TNFa levels do not increase at 24 or 72 h after TBI, when macrophage infiltration occurs, suggests that these cells are not major sources of post-traumatic TNFa increases.
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Beneficial effects of monomeric, soluble, TNFR II receptors have been observed after i.v. administration following closed head injury, where they improved neurological recovery and prevented neuronal damage ŽShohami et al., 1996.. The current data represent important additional evidence, utilizing a different model and selective antagonist, that TNFa contributes to functional impairment after TBI. sTNFR:Fc was quite effective i.c.v., but, even over a range of doses, failed to improve outcome when given i.v. This finding may reflect differences between monomeric and dimeric soluble receptor constructs. Because TNFa primarily exists in trimeric form, the dimeric structure of sTNFR:Fc results in substantially greater affinity for TNFa than monomeric TNFR II receptors ŽPeppel et al., 1991; Schoenfeld et al., 1991.. sTNFR:Fc is also a more potent functional antagonist of TNFa activity ŽMohler et al., 1993.. However, the Fc backbone that helps confer these properties, along with the additional receptor moiety, greatly increases the size of this molecule. This may limit its ability to cross the blood–brain barrier, presumably even when compromised. Our data, in concurrence with those of others, indicates that the presence of TNFa in the early period after TBI is detrimental to functional outcome. Yet, explanations as to why this is so are not obvious, especially when viewed in light of growing evidence showing beneficial effects of TNFa after acute CNS injury in transgenic models ŽBruce et al., 1996; Scherbel et al., 1997; Sullivan et al., 1997., and also in mechanistic studies ŽMattson et al., 1995.. There are several possible explanations for these disparate findings. First, the early rise in TNFa that occurs after TBI may be detrimental, because it increases levels beyond that needed for cellular protection. Thus, TNFa produced by many individual neurons, as part of a local autocrine or paracrine protective response, may accumulate regionally and ultimately reach cytotoxic levels. The observation that TNFa is elevated in CSF after TBI ŽRoss et al., 1994. supports the notion that a significant overproduction of TNFa may exist. Neurons are relatively resistant to TNFa cytotoxicity ŽCheng et al., 1994; D’Souza et al., 1995.; however, actual local concentrations that occur after TBI, which are unknown, may be neurotoxic. Moreover, other cell-types, such as oligodendrocytes and endothelial cells, are differentially sensitive to the toxic effects of TNFa ŽD’Souza et al., 1995; de Vries et al., 1996; Duchini et al., 1996., and under conditions of overproduction, damage to these cells may indirectly lead to tissue loss and worsened outcome. Second, TNFa may subserve differential physiological roles over time after injury. TNFa knock-out mice performed better on neurological tests given in the first 48 h after TBI than did their respective controls, even though weeks later, this same group subsequently performed significantly worse ŽScherbel et al., 1997.. Thus, the actions of TNFa may be detrimental during the acute phase of injury, but beneficial in later phases of the secondary injury process. Third, cellular effects of TNFa are likely
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to be altered by other ongoing secondary injury events. For example, TNFa enhances the toxicity of excitatory amino acids ŽChao and Hu, 1994., which are also increased early after TBI ŽFaden et al., 1989.. Also, some cell survival strategies induced by TNFa depend on protein synthesis ŽCheng et al., 1994; Mattson et al., 1995; Wallach, 1997.. Because synthetic processes are inhibited in cells stressed or damaged by CNS injury ŽBodsch et al., 1985; Thilmann et al., 1987., it is possible that such protective schemes, potentially initiated by TNFa , cannot be completed. Consistent with this idea, most of the known protective effects of TNFa have been observed in pre-treatment paradigms ŽCheng et al., 1994; Nawashiro et al., 1997.. Lastly, the cellular actions of TNFa are complex and subject to divergent regulation. Thus, identical cell types may respond differently to TNFa , depending on such influencing factors as activated receptor subtype ŽSipe et al., 1996; Shen et al., 1997., receptor proportion ŽBotchkina et al., 1997., and cross-talk between other intracellular pathways ŽYuan, 1997.. The effect of TBI on many of these variables is, as yet, largely unknown. In summary, the present data show that early, neuronal production of TNFa contributes to neurological dysfunction after TBI. This suggests that although TNFa has many diverse effects, it primarily subserves a pathophysiological role in the secondary injury processes that occur soon after CNS trauma.
Acknowledgements All procedures involving live animals were approved by the Georgetown University Animal Care and Use Committee and conducted according to the principles set forth in the Guide for the Care and Use of Laboratory Animals, prepared by the Committee on the Care and Use of Laboratory Animals of the Institute of Laboratory Resources, National Research Council ŽDHEW Pub aNIH 85-232985.. We thank Randi Goodnight and Susan Wishner for technical assistance. These studies were supported by grant aR49CCR3-6634-07 from the Centers for Disease Control to A.I. Faden. S.M. Knoblach was supported in part by NIH 5T32HD07459.
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