Intrathecal cyclosporin prolongs survival of late-stage ALS mice

Brain Research 894 (2001) 327–331 www.elsevier.com / locate / bres

Short communication

Intrathecal cyclosporin prolongs survival of late-stage ALS mice Marcus Keep a

a,c ,*,1

´ a,b,c , Keith S.K. Fong a , Katalin Csiszar a , Eskil Elmer

Laboratory of Matrix Pathobiology, Pacific Biomedical Research Center, University of Hawaii, 1960 East-West Road, Honolulu, HI 96822, USA b Laboratory for Experimental Brain Research, Wallenberg Neuroscience Center, Lund University Hospital, SE-221 85 Lund, Sweden c Maas BiolAB, LLC, 2228 Liliha Street, Suite 208, Honolulu, HI 96817, USA Accepted 22 December 2000

Abstract Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by upper and lower motor neuron death with ascending paralysis leading to death. In a transgenic mouse model of ALS (SOD1-G93A) weakness appears at 3 months of age, and because of progressive paralysis leads to death by 5 months. Cyclosporin A (CsA) is well known, for its extracerebral effect, as an immunosuppressant in organ transplantation. When able to access the brain, CsA is an effective neuroprotective agent mainly due to its protection of mitochondria through inhibition of the mitochondrial permeability transition. CsA does not cross the intact blood–brain barrier and was in the present study delivered to the brain through an infusion into the lateral cerebral ventricle. Injections started at the onset of late disease when weakness of the hindlimbs was apparent. CsA treatment prolonged the survival of ALS transgenic mice as compared to vehicle-treated controls. This finding implicates mitochondrial function in ALS and may have significance for human disease.  2001 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Neuromuscular diseases Keywords: Amyotrophic lateral sclerosis; Neurodegeneration; SOD1-G93A transgenic mice; Mitochondrial permeability transition; Intrathecal cyclosporin A; Neuroprotection

Amyotrophic lateral sclerosis (ALS) is a motor neuron degenerative disease characterized by progressive loss of upper motor neurons in the motor cortex and lower motor neurons in the brain stem and spinal cord. It is the most common of the human motor neuron diseases, and has only limited treatment options. The synchronous loss of upper and lower motor neurons creates a picture of non-spastic paralysis with skeletal muscle wasting. Ascending weakness results in paralysis. Death is usually seen in 3–5 years from loss of motor cranial nerves coordinating swallowing and breathing [46]. Ninety percent of the disease is sporadic and the 10% that is familial (FALS) has addition-

1

URL: http: / / lmp.biomed.hawaii.edu / people / profiles / profile marcus ] ] keep.html. *Corresponding author. Laboratory of Matrix Pathobiology, Pacific Biomedical Research Center, University of Hawaii, 1960 East-West Road, Honolulu, HI 96822, USA. Tel.: 11-808-547-6940; fax: 11-808-5476952. E-mail address: [email protected] (M. Keep).

al pathological features [17]. Twenty percent of FALS cases are associated with a Cu / Zn superoxide dismutase enzyme (Cu / Zn-SOD) mutation that has autosomal dominant inheritance [7]. This cytosolic metalloenzyme catalyzes superoxide conversion to hydrogen peroxide [6]. FALS sometimes manifests at a younger age, with a more rapid course to death compared to the sporadic form. While it is presently not known how the mutation causes motor neuron death, it has been attributed to a ‘gain of adverse function’, such as the overproduction of hydroxyl radicals [4] with increased cellular oxidative stress [1,10,31]. A transgenic mouse strain overexpressing the human SOD1 gene containing a Gly-93→Ala mutation (G93A) serves as a model for human ALS. These mice develop a progressive, ascending paralysis similar to human ALS. Disease usually starts with tremors and ends with hindlimb proximal atrophy [16] and complete paralysis of both hindlimbs at the time of euthanasia. Dal Canto and Gurney [8] describe early vacuolar degeneration of neurons and processes of the anterior horn cells, with late-stage Lewy

0006-8993 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 01 )02012-1

328

M. Keep et al. / Brain Research 894 (2001) 327 – 331

body-like inclusions, cell loss and anterior horn atrophy. Mitochondria are severely affected, with dilation of cristae progressing at late stages to fragmentation. In the G93A ALS model, various antioxidant and neuroprotective compounds including vitamin E, riluzole, and gabapentine [15] have been tested with varied effectiveness. Cyclosporin A (CsA) is a well-known immunosuppressive agent and exerts this effect by binding to intracellular cyclophilins, together inhibiting the calcium-dependent serine / threonine protein phosphatase calcineurin within immune cells. It has been demonstrated that cyclophilins are highly enriched in neurons, especially the brainstem motor nuclei [25]. CsA has recently been shown to exert potent neuroprotection in animal models of global ischemia [30,44,45], focal ischemia [24,33,47], and traumatic brain injury [5,35,36,40,41] when it can cross the blood–brain barrier. It is well established that CsA prevents the assembly of the mitochondrial permeability transition (mPT) pore (which is formed under certain conditions when mitochondria are loaded with calcium) [23,27]. CsA thereby stabilizes neuronal mitochondrial membranes and function and prevents release of apoptogenic factors and resulting cell death. Thus, the neuroprotective effect of CsA is unrelated to its action on calcineurin within immune cells. This is evident from experiments with a non-immunosuppressive analogue of CsA, which is an effective neuroprotectant, and supported by the lack of effect of the calcineurin inhibitor, and immunosuppressive agent, FK506 in hypoglycemic coma and cortical impact models [11,33,40]. Since FALS seems to involve mitochondrial function, the hypothesis that CsA as a mitochondrial protectant might slow progression of motor neuron death and paralysis and prolong survival was conceived. One study of cell cultures transfected with mutant SOD demonstrated increased cell death when CsA was added to the culture medium [26]. However, the transfected cells were hippocampal pyramidal neurons and therefore the results are difficult to extrapolate to in vivo administration of CsA and degenerating motor neurons in FALS mice. Surprisingly, lipophilic CsA does not cross the blood–brain barrier at immunosuppressive doses. It is actively excluded from the brain by the capillary endothelial ATP-dependent P-glycoprotein transporter [3,43]. From several known ways to deliver CsA across the blood–brain barrier into the brain, we selected intracerebroventricular injections through an implanted cannula. Humans with a diagnosis of ALS within a few years advance to late-stage disease. The objective of the present study was therefore to evaluate the effect of CsA on late-stage ALS mice progressing from limb weakness to paralysis, and survival. SOD1 transgenic mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA). The strain designated B6SJL-TgN(SOD1-G93A)2Gur was maintained by breeding homozygous carriers to B6SJLF1 hybrids. Standard polymerase chain reaction (PCR) protocol using two sets

of primers was used as confirmatory genotyping [14]. All procedures complied with federal guidelines and the experimental protocol was approved by the University of Hawaii Institutional Animal Care and Use Committee. Transgenic mice at 3 months of age were anesthetized with pentobarbital (60 mg / kg, i.p.) and placed in a Kopf stereotaxic frame. A 24 gauge guide cannula (Plastics One, Roanoke, VA, USA) was implanted into the right lateral ventricle (coordinates from bregma AP50.0 mm, ML51 1.0 mm, DV522.5 mm; toothbar50.0 mm) in all animals and fixed to the skull with a jeweler’s screw and dental cement. Following surgery the mice were placed in individual cages with free access to water and food on a 12-h light–dark cycle. Mice were examined every 2 days for irregularity of gait, limping or dragging of hindlimbs, and weighed. Spasticity was sought by observing tonic elevation of the tail either spontaneously or on stroking. Strength and agility of the hindlimbs were tested by the ability to grasp the cage top bars and walk upside down. Loss of the ability to use hindlimbs to grasp the cage bars while upside-down was the diagnosis point for late-stage disease. Diagnosis was confirmed by a second examiner. Following diagnosis mice were examined daily and randomized to CsA (n57) and vehicle (n55) infusions every other day. Four additional mice served as non-operated controls. A 5-ml (25 mg CsA, 0.5% CsA in 20% soybean oil and lecithin, oil-in-water emulsion) volume (CicloMulsion, Germany) was injected intrathecally with Hamilton syringe and a 30 gauge inner cannula. Three mice were inadvertently given CsA on 2 consecutive days (instead of every 2 days), thereafter resuming every other day dosing. One of these mice died 2 days later from presumed overdose (day 3 following diagnosis) and was excluded. Paralysis of both hindlimbs or inability to eat with resultant 25% mass loss was defined as the endpoint (time of death) and the animals were euthanized with an overdose of pentobarbital and transcardially perfused in buffered 4% paraformaldehyde (PFA). Brains were removed and postfixed in 4% PFA. The implanted cannula was found to extend to the lateral ventricle in all operated animals. One-way analysis of variance (ANOVA) followed by Scheffe´ post-hoc test was used to compare time to endpoint between the groups. Significance was set at P, 0.05 and data are presented as mean6S.E.M. Mice receiving intrathecal vehicle infusions reached endpoint 11.861.7 days after diagnosis with a range of 8 through 18 days (Fig. 1) and behaved and progressed neurologically in a manner identical to non-treated control ALS mice which reached endpoint after 10.261.6 days (Fig. 1). Hindlimbs slowly became paralyzed and demonstrated markedly increased tone. Forepaws weakened, but did not become paralyzed. The spine developed a kyphotic arch and tail tone dramatically increased. Stroking the tail induced reflex 908 elevation of the tail. Mice were completely alert with normal head movements and ate from a pellet placed in their forepaws up until the time of

M. Keep et al. / Brain Research 894 (2001) 327 – 331

Fig. 1. Survival times following diagnosis of late-stage ALS in mice carrying the SOD1-G93A mutant gene. Mice were diagnosed as late-stage when they were no longer able to use their hindlimbs to climb upside down. At diagnosis intracerebroventricular infusions of cyclosporin A (CsA, n56) or its vehicle (n55) were started. Control mice (n54) were only subjected to examination of hindlimb strength and agility and were not subjected to surgery or treatment. Endpoint was paralysis of both hindlimbs. Group mean values are represented by horizontal bars. *P, ´ 0.05; one-way ANOVA followed by post-hoc Scheffe.

euthanasia. Stool would collect in a pile because of immobility on the last day prior to euthanasia. Mice receiving CsA infusions behaved and progressed in a markedly different way. Two mice displayed asymmetry of motion. The left side became more robust and active. The two mice leaned with the right side against the cage wall and moved vigorously in a counterclockwise direction. When placed with the left side against the wall, the two mice would fall onto their right side and rotate in circles propelled with the left limbs until able to again position their weaker side against the cage wall to resume propulsion in a counterclockwise fashion. All the CsA treated mice became gradually weaker with a pattern different from the vehicle-treated mice. The hindlimbs weakened and finally paralyzed, but did not increase in tone or spasticity. The spinal column did not become arched. The tail became less active, but remained supple without increased tone and not becoming erect, even with stroking. Mice receiving CsA infusions lived 24.264.1 days after diagnosis with a range of 16 through 37 days (Fig. 1). Two mice that reached endpoint at 17 and 19 days did so without paralysis. They seemed lethargic, but arousable. The lethargy led to decreased feeding and drinking activating the excess mass loss criteria for

329

euthanasia. This lethargy was not seen in non-treated or vehicle mice. One CsA-treated mouse was euthanized at 37 days when the skull cannula dislodged, even though it was still active and had not yet reached paralysis or other criteria for euthanasia. Ninety percent of people with ALS have the sporadic disease and thus cannot be pre-diagnosed as potentially those with FALS might be. Thus it is not possible for them to be ‘pretreated’ with a medication before they develop weakness. Drugs which have been shown to slow mouse disease when given a month before weakness develops, and increases total lifespan by 27 days for caspase inhibitors [29], 26 days for 2% creatine [20], 10 days for penicillamine [18] or 9 days for carboxyfullerenes [9], are of uncertain utility for humans already diagnosed with clinical disease but may shed important light on the pathogenesis of ALS. Using the hypothesis that ALS is an autoimmune disease, a human double-blind study in 1988 attempted to treat ALS with CsA. Patients received 10 mg / kg systemic CsA for up to 40 weeks. The same motor decline was seen in both placebo and CsA-treated patients [2,42]. The study conclusion was that ALS is not influenced by immunosuppression. Any possible direct neuroprotective effect that CsA might have is not revealed by that study because CsA does not cross the intact blood–brain barrier and was not able to reach the motor neurons of those patients [28]. The intrathecal dose of CsA given here (25 mg every other day) seems to be tolerated by most ALS transgenic mice. The dose given represents approximately a 18 mg / kg / day CsA dose seen by the craniospinal neuroaxis. One mouse in three that was accidentally given 25 mg doses on 2 consecutive days died shortly thereafter. Of interest, the two mice that survived went on to live the longest of the mice, both 37 days. Two other CsA mice succumbed at days 17 and 19, not from paralysis but from not eating or drinking, which might be related to the administration of CsA. We have some indications in early pilot studies that higher doses of intrathecal CsA makes animals initially hyperactive, then apparently dazed and docile, uninterested in eating or drinking (unpublished observations). The late-stage disease was chosen, from the day when the mice could no longer grasp the cage roofbars with hindpaws while hanging upside down. From this point, the normal course seen in other studies is rapid progression to hindlimb paralysis in 1161 days [37] or 1262 days [12]. Our vehicle mice lived 11.862 days, demonstrating that surgery and intrathecal lipid emulsion injections did not alter survival time. CsA treated mice lived 24.264 days, which is 12 days longer than vehicle controls. This survival at even latestage administration is comparable in length to extensions in survival seen with long-term riluzole pre-treatment [15]. FALS mouse mitochondria show the prominent pathological features of microvacuolarization, swelling and distortion [13]. A significant increase in this vacuolariza-

330

M. Keep et al. / Brain Research 894 (2001) 327 – 331

tion concurrent with rapid motor decline suggests that mitochondria are directly linked to motor neuron death [21]. Bcl-2 is a protein on the outer mitochondrial membrane. It has been demonstrated to inhibit apoptosis and necrosis through increasing resistance to mPT formation and release of apoptogenic factors [19,34,38,39]. Overexpression of Bcl-2 prolongs survival by 12.5% in FALS mice [22]. Furthermore, caspases have been implicated in mouse ALS [32] and Li et al. [29] found that intrathecal infusion at a pre-clinical phase of a caspase inhibitor (zVAD-fmk) prolonged total survival by 27 days. More work is in needed to characterize the therapeutic and toxic ranges for intrathecal CsA administration. An important future task is to carefully evaluate possible CsA effects on ALS transgenic mouse spinal motor neuron loss at defined stages of the disease. In conclusion, CsA treatment positively influences the survival of late-stage ALS transgenic mice and implicates mitochondrial function in ALS.

[9]

[10]

[11]

[12] [13] [14] [15]

[16]

Acknowledgements This study was supported by the Victoria S. and Bradley L. Geist Foundation, the Restorative Neurosurgery Foundation and Maas BiolAB, LLC’s. Maas BiolAB, LLCs’ intellectual property includes the use of cyclosporin for neurological indications. Cyclosporin A in lipid emulsion was kindly provided by CicloMulsion, Germany.

[17]

[18]

[19]

References [1] P.K. Andrus, T.J. Fleck, M.E. Gurney, E.D. Hall, Protein oxidative damage in a transgenic mouse model of familial amyotrophic lateral sclerosis, J. Neurochem. 71 (1998) 2041–2048. [2] S.H. Appel, S.S. Stewart, V. Appel, Y. Harati, W. Mietlowski, W. Weiss, G.W. Belendiuk, A double-blind study of the effectiveness of cyclosporine in amyotrophic lateral sclerosis, Arch. Neurol. 45 (1988) 381–386. [3] D.J. Begley, L.K. Squires, B.V. Zlokovic, D.M. Mitrovic, C.C. Hughes, P.A. Revest, J. Greenwood, Permeability of the blood brain barrier to the immunosuppressive cyclic peptide cyclosporin A, J. Neurochem. 55 (1990) 1222–1230. [4] M.B. Bogdanov, L.E. Ramos, Z. Xu, M.F. Beal, Elevated ‘hydroxyl radical’ generation in vivo in an animal model of amyotrophic lateral sclerosis, J. Neurochem. 71 (1998) 1321–1324. [5] A. Buki, D.O. Okonkwo, J.T. Povlishock, Postinjury cyclosporin A administration limits axonal damage and disconnection in traumatic brain injury, J. Neurotrauma 16 (1999) 511–521. [6] J.D. Crapo, T. Oury, C. Rabouille, J.W. Slot, L.Y. Chang, Copper, zinc superoxide dismutase is primarily a cytosolic protein in human cells, Proc. Natl. Acad. Sci. USA 89 (1992) 10405–10409. [7] M.E. Cudkowicz, D. McKenna-Yasek, P.E. Sapp, W. Chin, B. Geller, D.L. Hayden, D.A. Schoenfeld, B.A. Hosler, H.R. Horvitz, R.H. Brown, Epidemiology of mutations in superoxide dismutase in amyotrophic lateral sclerosis, Ann. Neurol. 41 (1997) 210–221. [8] M.C. Dal Canto, M.E. Gurney, Neuropathological changes in two lines of mice carrying a transgene for mutant human Cu, Zn SOD,

[20]

[21]

[22]

[23]

[24]

[25]

[26]

and in mice overexpressing wild type human SOD: a model of familial amyotrophic lateral sclerosis (FALS), Brain Res. 676 (1995) 25–40. L.L. Dugan, D.M. Turetsky, C. Du, D. Lobner, M. Wheeler, C.R. Almli, C.K. Shen, T.Y. Luh, D.W. Choi, T.S. Lin, Carboxyfullerenes as neuroprotective agents, Proc. Natl. Acad. Sci. USA 94 (1997) 9434–9439. R.J. Ferrante, S.E. Browne, L.A. Shinobu, A.C. Bowling, M.J. Baik, U. MacGarvey, N.W. Kowall, R.H. Brown Jr., M.F. Beal, Evidence of increased oxidative damage in both sporadic and familial amyotrophic lateral sclerosis, J. Neurochem. 69 (1997) 2064–2074. H. Friberg, M. Ferrand-Drake, F. Bengtsson, A.P. Halestrap, T. Wieloch, Cyclosporin A, but not FK 506, protects mitochondria and neurons against hypoglycemic damage and implicates the mitochondrial permeability transition in cell death, J. Neurosci. 18 (1998) 5151–5159. R.M. Friedlander, R.H. Brown, V. Gagliardini, J. Wang, J. Yuan, Inhibition of ICE slows ALS in mice, Nature 388 (1997) 31. M.E. Gurney, Transgenic-mouse model of amyotrophic lateral sclerosis, New Engl. J. Med. 331 (1994) 1721–1722. M.E. Gurney, Transgenic animal models of familial amyotrophic lateral sclerosis, J. Neurol. 244 (Suppl. 2) (1997) S15–S20. M.E. Gurney, F.B. Cutting, P. Zhai, A. Doble, C.P. Taylor, P.K. Andrus, E.D. Hall, Benefit of vitamin E, riluzole, and gabapentin in a transgenic model of familial amyotrophic lateral sclerosis, Ann. Neurol. 39 (1996) 147–157. M.E. Gurney, H. Pu, A.Y. Chiu, M.C. Dal Canto, C.Y. Polchow, D.D. Alexander, J. Caliendo, A. Hentati, Y.W. Kwon, H.X. Deng et al., Motor neuron degeneration in mice that express a human Cu, Zn superoxide dismutase mutation, Science 264 (1994) 1772–1775. A. Hirano, L.T. Kurland, G.P. Sayre, Familial amyotrophic lateral sclerosis. A subgroup characterized by posterior and spinocerebellar tract involvement and hyaline inclusions in the anterior horn cells, Arch. Neurol. 16 (1967) 232–243. A.F. Hottinger, E.G. Fine, M.E. Gurney, A.D. Zurn, P. Aebischer, The copper chelator D-penicillamine delays onset of disease and extends survival in a transgenic mouse model of familial amyotrophic lateral sclerosis, Eur. J. Neurosci. 9 (1997) 1548–1551. S. Kharbanda, P. Pandey, L. Schofield, S. Israels, R. Roncinske, K. Yoshida, A. Bharti, Z.M. Yuan, S. Saxena, R. Weichselbaum, C. Nalin, D. Kufe, Role for Bcl-xL as an inhibitor of cytosolic cytochrome C accumulation in DNA damage-induced apoptosis, Proc. Natl. Acad. Sci. USA 94 (1997) 6939–6942. P. Klivenyi, R.J. Ferrante, R.T. Matthews, M.B. Bogdanov, A.M. Klein, O.A. Andreassen, G. Mueller, M. Wermer, R. KaddurahDaouk, M.F. Beal, Neuroprotective effects of creatine in a transgenic animal model of amyotrophic lateral sclerosis, Nat. Med. 5 (1999) 347–350. J. Kong, Z. Xu, Massive mitochondrial degeneration in motor neurons triggers the onset of amyotrophic lateral sclerosis in mice expressing a mutant SOD1, J. Neurosci. 18 (1998) 3241–3250. V. Kostic, V. Jackson-Lewis, F. de Bilbao, M. Dubois-Dauphin, S. Przedborski, Bcl-2: prolonging life in a transgenic mouse model of familial amyotrophic lateral sclerosis, Science 277 (1997) 559–562. B.S. Kristal, J.M. Dubinsky, Mitochondrial permeability transition in the central nervous system: induction by calcium cycling-dependent and -independent pathways, J. Neurochem. 69 (1997) 524–538. ¨ The immunosuppressants S. Kuroda, S. Janelidze, B.K. Siesjo, cyclosporin A and FK506 equally ameliorate brain damage due to 30-min middle cerebral artery occlusion in hyperglycemic rats, Brain Res. 835 (1999) 148–153. R.P. Lad, M.A. Smith, D.C. Hilt, Molecular cloning and regional distribution of rat brain cyclophilin, Mol. Brain Res. 9 (1991) 239–244. J.P. Lee, H.C. Palfrey, V.P. Bindokas, G.D. Ghadge, L. Ma, R.J. Miller, R.P. Roos, The role of immunophilins in mutant superoxide dismutase-1 linked familial amyotrophic lateral sclerosis, Proc. Natl. Acad. Sci. USA 96 (1999) 3251–3256.

M. Keep et al. / Brain Research 894 (2001) 327 – 331 [27] J.J. Lemasters, V. Necrapoptosis and the mitochondrial permeability transition: shared pathways to necrosis and apoptosis, Am. J. Physiol. 276 (1999) G1–6. [28] G.L. Lensmeyer, D.A. Wiebe, I.H. Carlson, R. Subramanian, Concentrations of cyclosporin A and its metabolites in human tissues postmortem, J. Anal. Toxicol. 15 (1991) 110–115. [29] M. Li, V.O. Ona, C. Guegan, M. Chen, V. Jackson-Lewis, L.J. Andrews, A.J. Olszewski, P.E. Stieg, J.P. Lee, S. Przedborski, R.M. Friedlander, Functional role of caspase-1 and caspase-3 in an ALS transgenic mouse model, Science 288 (2000) 335–339. ´ B.K. Siesjo, ¨ Amelioration by [30] P.A. Li, H. Uchino, E. Elmer, cyclosporin A of brain damage following 5 or 10 min of ischemia in rats subjected to preischemic hyperglycemia, Brain Res. 753 (1997) 133–140. [31] D. Liu, J. Wen, J. Liu, L. Li, The roles of free radicals in amyotrophic lateral sclerosis: reactive oxygen species and elevated oxidation of protein, DNA, and membrane phospholipids, FASEB J. 13 (1999) 2318–2328. [32] L.J. Martin, A.C. Price, A. Kaiser, A.Y. Shaikh, Z. Liu, Mechanisms for neuronal degeneration in amyotrophic lateral sclerosis and in models of motor neuron death, Int. J. Mol. Med. 5 (2000) 3–13. [33] S. Matsumoto, H. Friberg, M. Ferrand-Drake, T. Wieloch, Blockade of the mitochondrial permeability transition pore diminishes infarct size in the rat after transient middle cerebral artery occlusion, J. Cereb. Blood Flow Metab. 19 (1999) 736–741. [34] R.G. Miller, New approaches to therapy of amyotrophic lateral sclerosis, West J. Med. 168 (1998) 262–263. [35] D.O. Okonkwo, A. Buki, R. Siman, J.T. Povlishock, Cyclosporin A limits calcium-induced axonal damage following traumatic brain injury, NeuroReport 10 (1999) 353–358. [36] D.O. Okonkwo, J.T. Povlishock, An intrathecal bolus of cyclosporin A before injury preserves mitochondrial integrity and attenuates axonal disruption in traumatic brain injury, J. Cereb. Blood Flow Metab. 19 (1999) 443–451.

331

[37] W.A. Pedersen, M.P. Mattson, No benefit of dietary restriction on disease onset or progression in amyotrophic lateral sclerosis Cu / Znsuperoxide dismutase mutant mice, Brain Res. 833 (1999) 117–120. [38] J.H. Prehn, V.P. Bindokas, C.J. Marcuccilli, S. Krajewski, J.C. Reed, R.J. Miller, Regulation of neuronal Bcl2 protein expression and calcium homeostasis by transforming growth factor type beta confers wide-ranging protection on rat hippocampal neurons, Proc. Natl. Acad. Sci. USA 91 (1994) 12599–12603. [39] J.C. Reed, Cytochrome c: can’t live with it – can’t live without it, Cell 91 (1997) 559–562. [40] S.W. Scheff, P.G. Sullivan, Cyclosporin A significantly ameliorates cortical damage following experimental traumatic brain injury in rodents, J. Neurotrauma 16 (1999) 783–792. [41] P.G. Sullivan, M. Thompson, S.W. Scheff, Continuous infusion of cyclosporin A postinjury significantly ameliorates cortical damage following traumatic brain injury, Exp. Neurol. 161 (2000) 631–637. [42] R.S. Tindall, Immunointervention with cyclosporin A in autoimmune neurological disorders, J. Autoimmun. 5 (Suppl. A) (1992) 301–313. [43] A. Tsuji, P-Glycoprotein-mediated efflux transport of anticancer drugs at the blood–brain barrier, Ther. Drug Monit. 20 (1998) 588–590. ´ K. Uchino, P.A. Li, Q.P. He, M.L. Smith, B.K. [44] H. Uchino, E. Elmer, ¨ Amelioration by cyclosporin A of brain damage in transient Siesjo, forebrain ischemia in the rat, Brain Res. 812 (1998) 216–226. ´ K. Uchino, O. Lindvall, B.K. Siesjo, ¨ Cyclos[45] H. Uchino, E. Elmer, porin A dramatically ameliorates CA1 hippocampal damage following transient forebrain ischaemia in the rat, Acta Physiol. Scand. 155 (1995) 469–471. [46] D.B. Williams, A.J. Windebank, Motor neuron disease (amyotrophic lateral sclerosis), Mayo Clin. Proc. 66 (1991) 54–82. ¨ Posttreatment with the immunosuppres[47] T. Yoshimoto, B.K. Siesjo, sant cyclosporin A in transient focal ischemia, Brain Res. 839 (1999) 283–291.