Journal of Cerebral Blood Flow and Metaholism 19:1329-1335, 1999 Published by Lippincott Williams & Wilkins, Inc., Philadelphia

Transplantation of Fetal Kidney Tissue Reduces Cerebral

Infarction Induced by Middle Cerebral Artery Ligation

Yung-Hsiao Chiang, Shinn-Zong Lin, *Cesario V. Borlongan, *Barry J. Hoffer,

*Marisela Morales, and *Yun Wang

Departments of Neurosurgery and Pharmacology, Tri-Service General Hospital National Defense Medical Center, Taipei,

Taiwan; and *National Institute on Drug Abuse, Baltimore, Maryland, US.A.

Summary: The authors, and others, have recently reported that intracerebral administration of glial cell line-deri ved neu­rotrophic factor (GDNF) or osteogenic protein-l protects against ischemia-induced injury in the cerebral cortex of adult rats. Because these trophic factors are highly expressed in the fetal, but not adult, kidney cortex, the possibility that transplan­tation of fetal kidney tissue could serve as a cellular reservoir for such molecules and protect against ischemic injury in ce­rebral cortex was examined. Fetal kidneys obtained from rat embryos at gestational day 16, and adult kidney cortex, were dissected and cut into small pieces. Adult male Sprague­Dawley rats were anesthetized with chloral hydrate and placed in a stereotactic apparatus. Kidney tissues were transplanted into three cortical areas adjacent to the right middle cerebral artery (MCA). Thirty minutes after grafting, the right MCA was transiently ligated for 90 minutes. Twenty-four hours after the onset of reperfusion, animals were evaluated behaviorally.

Cerebral ischemia and reperfusion can induce irrevers­ible damage to the brain. Several different classes of drugs have been proposed for pre- and postischemic treatment, but none have demonstrated clear-cut efficacy in clinical trials. Recent studies have indicated that ex­pression of neurotrophic factors is associated with brain injury (Hoffer and Olson, 1 997; Liberatore et ai., 1 997). Systemic administration of kainate or pilocarpine in­duces glial cell line-derived neurotrophic factor (GDNF) (Humpel et ai., 1 994; Schmidt-Kastner et ai., 1994) and GDNF receptor-alpha (GFR-al) expression (Reeben et ai., 1 998). Similarly, GDNF mRNA expression in the

Received December 31, 1998; final revision received April 27, 1999;

May 26. 1999.

This is a U.S. Government work. There are no restrictions on its use. This work was supported by the National Institute on Drug Abuse. Address correspondence to Dr. Yun Wang, National Institute on

Drug Abuse, 5500 Nathan Shock Drive, Baltimore, MD 21224, U.S.A. Abbreviations used: GDNF, glial cell line-derived neurotrophic fac­

tor; MeA, middle cerebral artery; PB, phosphate buffer.

1329

It was found that the stroke animals that received adult kidney transplantation developed motor imbalance. However, animals that received fetal kidney grafts showed significant behavioral improvement. Animals were later sacrificed and brains were removed for triphenyltetrazolium chloride staining. Pax-2 im­munostaining. and GDNF mRNA expression. It was noted that transplantation of fetal kidney but not adult kidney tissue greatly reduced the volume of infarction in the cerebral cortex. Fetal kidney grafts showed Pax-2 immunoreactivity and GDNF mRNA in the host cerebral cortex. In contrast, GDNF mRNA expression was not found in the adult kidney grafts. Taken together, our data indicate that fetal kidney transplantation re­duces ischemialreperfusion-induced cortical infarction and be­havioral deficits in adult rats, and that such tissue grafts could serve as an unique cellular reservoir for trophic factor applica­tion to the brain. Key Words: Ischemia-Trophic factors­glial cell line-derived neurotrophic factor (GDNF)-Kidney.

striatum was increased in animals treated with I-methyl-4-phenyl-l,2,3,6-tetrahydropyridine for 7 days (Tang et aI., 1 998) or within 6 hours after mechanical injury to adult mouse striatum (Liberatore et ai., 1 997). In comple­mentary fashion, pretreatment with these neurotrophic factors often decreases the effect of insults. Pretreatment with GDNF reduces 6-0HDA (Hoffer et ai., 1 994; Bo­wenkamp et ai., 1 995), I-methyl-4-phenyl-l ,2,3,6- tetra­hydropyridine (Tomac et aI., 1 995), or axotomy-induced neuronal damage in the nigrostriatal dopaminergic path­way (Beck et aI., 1 995). GDNF also diminishes damage produced by kainate-induced tonic-clonic convulsions (Martin et ai., 1 995). It is thus possible that these trophic factors are endogenous neuroprotective agents and that they are upregulated after brain insults to help rescue neuronal circuitry.

Recent studies have shown that hypoxia or brain isch­emia induces trophic factor expression just as chemical insults do. GDNF, a protein in the TGF-j3 superfamily, is upregulated after cerebral ischemiaireperfusion induced by a MCA occlusion (Abe and Hayashi, 1997). We and

1330 Y-H. CHIANG ET AL.

others have recently reported that intracerebral adminis­tration or topical application of GDNF decreases isch­emia-induced brain infarction (Wang et a!., 1 997; Kitagawa et a!., 1 998b). GDNF decreases MCA occlu­sion-induced brain edema, the density of TUNEL (+) neurons in the ischemic cortex (Abe et a!., 1 997; Kitagawa et a!., 1 998a,b), and immunoreactivity of caspases-l and 3 (Kitagawa et a!., 1 998b). We also found that ischemia and reperfusion-induced nitric oxide re­lease is attenuated by pretreatment with GDNF (Wang et aI., 1 997). Taken together, these data suggest that GDNF may reduce ischemic insults by attenuating apoptosis and/or necrosis. We and others recently have also re­ported that osteogenic protein-I, another member of the TGF-� superfamily, is neuroprotective (Lin et a!., 1999). Thus, it is possible that GDNF, OP-I, and other TGF-� superfamily proteins are endogenous neuroprotective agents in stroke.

In the absence of perturbations, little GDNF is ex­pressed in adult neuronal tissue (Stromberg et aI., 1 993). GDNF, however, can be found in fetal neural and non­neuronal tissue. The outer mesenchyme of developing metanephric kidney contains a particularly strong GDNF mRNA signal, which peaks at a gestational age of 1 6 to 21 days (Nosrat et a!., 1 996). GDNF family a-receptors, such as <aFRal, are also highly expressed and coex­pressed with Ret mRNA in the developing kidney (Yu et aI., 1 998). GDNF and GFRal in fetal kidney are criti­cally involved in the development of the renal cortex. It has been found that mice which are deficient in GDNF have no kidneys (Moore et aI., 1 996; Pichel et aI., 1 996). Most of the animals that lacked GFRa I also had com­plete bilateral renal agenesis and ureteral deficits (Ca­calano et aI., 1 998). Other trophic factors in the TGF-� superfamily, such as OP-l (Dudley and Robertson, 1 997), neurturin (Widenfalk et aI., 1 997), and TGF-� itself (Choi et aI., 1 997; Basile and Hammerman, 1 998) are all highly expressed in the fetal kidney similar to GDNF.

Because these fetal kidney-containing trophic factors have all been shown to be neuroprotective, and because they may synergistically interact with other trophic fac­tors in the brain, we examined the possibility that graft­ing of fetal kidney may elicit neuroprotection during ischemia. As adult kidney manifests much lower expres­sion levels of these factors, such tissue was utilized for control grafts, in addition to standard vehicle controls.

METHODS

Animals A total of 32 adult male Sprague-Dawley rats (body weight,

>300 g) were used for kidney transplantation. The animals were divided into three groups for behavioral and infarction assays: 1) Fetal kidney transplantation group, which received fetal kid­ney grafts (n = 7). 2) Adult kidney transplantation group,

J Cereb Blood Flow Metab, Vol. 19, No. 12, 1999

which received grafts from adult kidney tissue (n = 8). 3) Vehicle controls, which received intracerebral Hank's balanced salt solution injection (30 fLL, n = 8). The other nine rats, six grafted with fetal tissue and three grafted with adult kidney tissue, were used for GDNF in situ hybridation and Pax-2 im­munostaining.

Transplantation Fetuses, at the gestational day of 16 to 17, were removed

from halothane-anesthetized pregnant rats by cesarean section. The kidneys were dissected, and the ureter and renal pelvis were removed. Kidneys were also obtained from adult rats. These animals were anesthetized with chloral hydrate. About 1 mm3 of cortical tissue from the adult kidney was dissected. Both adult and fetal kidney tissues were transferred into cold Hank's balanced salt solution after three washes with cold sa­line. Kidney tissues were cut into a slurry before transplanta­tion.

Each recipient rat received one fetal kidney or 9 to 12 fLL of adult kidney tissue for transplantation, which was further di­vided into three parts. The recipient rats were anesthetized with chloral hydrate (400 mg/kg, intraperitoneally). The kidney slurry, each about 3 to 4 fLl, was transplanted through a glass micropipette into three cortical areas adjacent to the right MCA, 2.0 to 3.0 mm below the dura (Fig. I). The approximate coordinates for these sites were: 1.0 to 2.0 mm anterior to the bregma and 3.5 to 4.0 mm lateral to the midline; 0.5 to 1.5 mm posterior to the bregma and 4.0 to 4.5 mm lateral to the mid­line;, and 4.0 to 4.5 mm posterior to the bregma and 5.5 to 6.0 mm lateral to the midline. The distance between transplant and major branches of MCA was approximately 1.0 to 1.5 mm. The speed of transplantation was 1 fLLlmin. The needle was re­tained in place for 5 minutes after each transplantation.

MCA ligation Thirty minutes after grafting, the anesthetized animals (chlo­

ral hydrate, 400 mg/kg, intraperitoneally) were subjected to cerebral ischemia. The ligation of the right MCA and bilateral common carotid arteries was performed using methods previ­ously described (Chen et aI., 1986). The bilateral common ca­rotid arteries were identified and isolated through a ventral midline cervical incision. The common carotid arteries were ligated with nontraumatic arterial clips. A craniotomy of about 4 mm2 was made in the right squamous bone. The right MCA was ligated with a 10-0 suture for 90 minutes. The craniotomy was then covered with a piece of gelfoam. Sutures were re­moved 90 minutes later because a 90-minute ligation induces maximal infarctions in rats of this age (Du et aI., 1996). After recovery from the anesthesia, the animals were returned to their home cages for a 24-hour reperfusion of the ischemic brain area.

Body temperature was monitored with a thermistor probe and maintained at 37°C with a heating pad during anesthesia. After recovery from the anesthesia, body temperature was fur­ther maintained at 37°C using a heat lamp.

Behavioral measurements Two behavioral assays were carried out after the 24-hour

reperfusion. In a qualitative postural reflex test modified from the Bederson behavioral assay (Bederson et aI., 1986), animals­were classified into two groups according to their neurologic deficits. (I) Behaviorally impaired: Rats keep their left fore­limb to the breast and extend the right forelimb straight or twist the upper half of their body when suspended 30 cm above the table or show decreased resistance to lateral push. (2) Behav­iorally normal: Rats extend both forelimbs straight and none of the observable deficits described in (1) are present.

FETAL KIDNEY GRAFTS AND ISCHEMIC STROKE DAMAGE 1331

A

Ligature

B

FIG. 1. Schematic diagrams of the sites of kidney grafts. MCA was ligated distally between the zygomatic arch and the first bifurcation of MCA above it. (A) Surface view showing fetal kid­ney tissue injections through a glass micropipette into right ce­rebral cortex, 2.0 to 3.0 mm below the dura. (8) Coronal sections showing that the approximate coordinates for these sites: (1) 1.0 to 2.0 mm anterior to the bregma and 3.5 to 4.0 mm lateral to the midline; (2) 0.5 to 1.5 mm posterior to the bregma and 4.0 to 4.5 mm lateral to the midline; and (3) 4.0 to 4.5 mm posterior to the bregma and 5.5 to 6.0 mm lateral to the midline. The distance between the transplants and major branches of MCA was about 1.0 to 1.5 mm. Scale bar: (A) 25 mm (8) 24 mm.

The stroke behavior was also quantitatively analyzed using a body swing test (Borlongan et aI., 1998). Rats were examined for lateral movements/turning when their bodies were sus­pended 30 em above the testing table by lifting their tails. The frequency of initial turning of head or upper body contralateral to the ischemic side was counted in 20 consecutive trials.

Triphenyltetrazolium chloride staining One day after reperfusion, animals were killed and perfused

intracardially with saline. The brain tissue was then removed, immersed in cold saline for 5 minutes, and sliced into 2.0-mm thick sections. The brain slices were incubated in a 2% triphe­nyltetrazolium chloride (Sigma) dissolved in normal saline for 30 minutes at 37°C and then transferred into a 5% formalde­hyde solution for fixation. The area of infarction in each slice was measured using a digital scanner and the Image Tools program (University of Texas Health Sciences Center in San Antonio) as described previously (Wang et aI., 1997; Lin et aI., 1999). The volume of infarction in each animal was obtained from the product of average slice thickness (2 mm) and sum of infarcted areas in all brain slices.

Pax-2 immunostaining and GDNF in situ hybridizations

Nine rats receiving fetal kidney tissue transplantation were perfused intracardially with 4% paraformaldehyde (l00 mLi min) in 0. 1 mol!L phosphate buffer (PB), pH 7.3. Brains were then removed from the skull, postfixed from 2 to 15 hours at 4°C, rinsed with PB, and sequentially transferred to 10%, 12%, and 15% sucrose solutions. They were then frozen on dry ice and sectioned on a cryostat to obtain coronal sections of 20 j.1m in thickness.

Pax -2 immunocytochemistry. Serial sections were first in­cubated in a blocking solution (PB supplemented with 4% bo­vine serum albumin and 0.3% Triton X-lOO) for 1 hour and then incubated with either blocking solution (control) or a well­characterized rabbit polycIonal anti-Pax 2 antibody raised against amino acids 188 to 385 (Babco, Richmond, CA, U.S.A.). Primary antibody was used at a final concentration of 5 to 20 j.1g/mL for 48 to 72 hours at 4°C. After rinsing 3 x 10 minutes in PB, sections were processed with an alkaline phos­phatase ABC kit (Vector, Burlingame, CA, U.S.A.). Material was incubated for 30 minutes at room temperature in a 1 :200 dilution of biotinylated anti-rabbit IgG. After rinsing with PB, sections were incubated with avidin-biotinylated alkaline phos­phatase for 30 minutes to 1 hour at room temperature. Samples were rinsed and the alkaline phosphatase reaction was devel­oped with nitroblue tetrazo1ium and 5-bromo-4-chloro-3-indolyl phosphate (Life Technologies, Gaithersburg, MD, U.S.A.). Controls consisted of omission of the primary anti­body.

GDNF in situ hybridization. In situ hybridization was per­formed as previously described (Morales and Bloom, 1997). After prehybridization, sections were hybridized at 55°C for 16 hours with eSS]-labeled sense or antisense single-stranded RNA probes (corresponding to GDNF nucIeotides 256 to 936 [Gen Bank Accession No. D 88264]) at 107 cprnlmL. Sections were treated with RNase A at 4 j.1g/mL at 37°C for 1 hour, washed in 1 x standard saline citrate, 50% formamide at 55°C for 1 hour, and in 0. 1 x standard saline citrate at 68°C for 30 minutes. Material was dehydrated and exposed on photographic film for 3 days.

Statistics Data were expressed using mean and standard deviation. One

way ANOVA and post hoc Newman-Keuls test were used for statistical analysis.

RESULTS

Behavioral measurements

Previous studies have shown that MeA ligation and reperfusion induce neurologic deficits in rats. We also found that 7 of 8 stroke rats pretreated with vehicle de­veloped postural imbalance as measured by the postural reflex test. Rats pretreated with fetal kidney tissue did not develop this behavioral abnormality. The incidence of postural imbalance in the fetal kidney transplant group was significantly lower than that in the vehicle control group (P < 0.05, Fisher Exact test). In the body swing test, we found that the frequency of swinging the body or head contralateral to the ischemic side was significantly higher in the stroke rats grafted with adult kidney tissue or injected with vehicle than in those pretreated with

J Cereb Blood Flow Metab, Vol. 19, No. 12, 1999

1332 Y-H. CHIANG ET AL.

fetal kidney (Fig. 2, P < 0.05, F = 1 3.0, df = 33, One way ANOVA + Newman-Keuls' test). There was no difference between the stroke rats that received fetal kid­ney grafts and nonlesioned rats in the swing test (Fig. 2).

Brain infarction

We and others have previously reported that ligation of the MeA for 90 minutes and reperfusion for 24 hours cause cortical infarction in rats. In the present study, we found that MeA ligation and reperfusion resulted in clear-cut infarction of the cortex in all the vehicle control animals studied (n = 8, Fig. 3). The volume (99.1 ± 44.2 mm3, mean ± SD) of infarction in vehicle control rats was not different from those in stroke rats pretreated with PB, or without any pretreatment, reported in our earlier studies (Wang et aI., 1 997).

Eight rats were transplanted with adult kidney tissue 30 minutes before MeA ligation. The volume (106.4 ± 42.4 mm3) of infarction was not different from vehicle control animals (Fig. 3). In contrast, the seven rats that received fetal kidney tissue transplants showed signifi­cantly smaller infarction volumes (39.5 ± 44.5 mm3) than vehicle control (99.1 ± 44.2 mm3) and adult kidney graft (1 06.4 ± 42.4 mm3) groups (Fig. 3, P < 0.05, F =

5.1 5, df = 22, one-way ANOVA + Newman-Keuls' test). The area of the largest infarction in a slice from each rat w�s also significantly diminished, from 1 5.6 ± 5.3 mm2 to 6.3 ± 5.6 mm2, after the fetal kidney trans­plantation (Fig. 3, P < 0.05, F = 7.89, df = 22, one-way

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Fetal Adult No No kidney kidney MeAo Graft

FIG.2. Motor imbalance is improved by fetal kidney tissue trans­plantation in stroke rats. In 20 consecutive trials, the frequency of swinging the upper body to the side contralateral to the lesion was significantly reduced in the stroke animals grafted with fetal kidney, compared to those pretreated with adult kidney cortex or vehicle (* P < 0.05, F = 13.0, df = 33, one-way ANOVA + New­man-Keuls' test). There is no difference between the lesioned rats that receive fetal grafts and nonlesioned animals (No MCAo).

J Cereb Blood Flow Metab. Vol. 19. No. 12. 1999

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B.

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c::::=J Fetal kidney lS§3 Adult kidney _ Vehicle

FIG. 3. Fetal kidney transplants attenuate cortical infarction in­duced by middle cerebral arterial (MCA) ligation. (A) Volume of infarction was significantly reduced in the fetal kidney transplant group as compared to the vehicle control and adult kidney tissue transplantation group (P < 0.05, df = 22, F = 5.15, one-way ANOVA + Newman Keuls' test). The volume of infarction equals 2 mm [thickness of the slice] x [sum of the infarction area in all brain slices (mm2). (B) The area of the largest infarction in a slice from a given rat was significantly diminished by fetal kidney trans­plants (P < 0.05, df = 22, F = 7.89, one-way ANOVA + Newman Keuls' test). (C) The number of infarcted slices per rat was re­duced by fetal kidney transplants (P < 0.05, df = 22, F = 4.64, one-way ANOVA + Newman-Keuls' test).

ANOVA + Newman-Keuls' test). Furthermore, the num­ber of infarcted slices in each rat was significantly re­duced (Fig. 3), from 5.0 ± 1 .5 slices/rat in vehicle-treated rats to 3.1 ± 2.0 slices/rat in the fetal kidney transplant rats (P < 0.05, F = 4.64, df = 22, one-way ANOV A + Newman-Keuls' test). Taken together, these data suggest that fetal kidney grafts diminish not only the volume but also the extent of infarction in the ischemic brain.

FETAL KIDNEY GRAFTS AND ISCHEMIC STROKE DAMAGE 1333

Pax·2 immunoreactivity of fetal kidney transplants

in the rat neocortex

We found that Pax-2 immunoreactivity was confined to the fetal kidney transplant (Fig. 4C). The average area of Pax-2 (+) staining was 0.42 mm2. No migration of grafted renal cells into adjacent cortical areas was seen. Sections where primary antibody was omitted showed no immunoreactivity of the fetal kidney transplant (Fig. 4D).

GDNF expression in the kidney grafts

Our unpublished observations have indicated that MeA ligation alone can induce upregulation of GDNF mRNA in the ischemic cortex. To eliminate confounds from the ischemic brain tissue, GDNF mRNA expression was studied in fetal and adult kidney tissue transplanted to the cortex of 6 nonischemic rats. We found that GDNF mRNA was expressed and restricted to the fetal trans­plant (Fig. 4A, arrow). In contrast, animals (n = 3) receiving adult kidney grafts did not show any GDNF mRNA expression in the transplants (Fig. 4B).

DISCUSSION

Previous studies have demonstrated that fetal cortical grafts can survive in the infarcted brain (Grabowski et aI., 1994; Tillotson et aI., 1 995; Jansen et aI., 1 997; Mattsson et aI., 1997). However, the extent of innerva­tion and behavioral recovery is controversial (Grabowski et aI., 1 995, 1 996; Sorenson et aI., 1 996) in the stroke animals. In the present study, we found that pretreatment with fetal kidney grafts in the cerebral cortex diminishes ischemia-induced behavioral deficits. It has been re­ported that the fetal kidney contains high levels of tro­phic factors and no neurons, as compared to fetal cortex. The fetal kidney graft-induced behavioral restoration

data suggest that trophic factors, rather than fetal neu­rons, are critical for neuroprotection in brain ischemia.

The decrease in infarction after fetal kidney transplan­tation is probably not attributable to kidney cells in the ischemic zone. Pax-2 immunostaining indicated that kid­ney tissue was limited to the graft site. The average vol­ume of Pax-2 (+) staining was 0.7 mm3 (0.4 mm2

x 0.6 mm x 3 sites). In contrast, the average reduction of in­farction volume was 59.6 mm3 in animals receiving fetal kidney transplants. Furthermore, marked infarction was also found in the animals with adult kidney grafts. Pre­vious studies have indicated that Pax-2 immunoreactivity is only present in the embryonic kidney which contains GDNF/GFRal moieties . The expression of Pax-2 in the fetal kidney may be regulated by GDNF mechanisms. Kidneys of El2.5 GFRal knock-out embryos do not express Pax-2 (Cacalano et aI., 1 998). We also found that GDNF mRNA is expressed only in the fetal kidney trans­plants. No GDNF mRNA activity was found in the adult kidney graft. We thus postulate that the decreased infarc­tion shown by triphenyltetrazolium chloride staining is not caused by the physical presence of renal cells but that diffusion of substances, possibly GDNF from the fetal kidney, underlies the neuroprotection seen here.

A recent study has indicated that fetal kidney has GDNF-like activity on midbrain dopamine neurons. Co­grafting of fetal kidney with ventral mesencephalic grafts in the 6-0HDA lesioned striatum increases outgrowth of DA fibers from these transplants (Granholm et aI., 1 998), also suggesting that fetal kidney tissue, like GDNF, has trophic activity for dopaminergic transplants.

Previous experiments have demonstrated that fetal kidney contains not only GDNF but also OP-l, neurturin, and TGF-[3 (Choi et aI., 1 997; Dudley and Robertson,

/' /

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FIG. 4. GDNF mRNA and Pax-2 immunoreactivity are present in the fetal kidney transplants in the rat neocortex. (A) Autoradiograph demonstrating the presence of GDNF mRNA restricted to the fetal kidney transplant (arrow). (8) No GDNF mRNA was found in the adult kidney graft (arrow) in the cortex. (C) Strong Pax-2 like immunoreactivity was confined to the fetal kidney transplant (arrows) when tissue was incubated with the anti-Pax antibody (0) Immunostaining control; note lack of immunoreactivity of the fetal kidney transplant (arrows) when primary antibody (anti-Pax) was omitted. Scale bar: A, B = 2.72 mm; C, 0 = 0.16 mm.

J Cereb Blood Flow Metab. Vol. 19. No. 12, 1999

1334 Y.-H. CHIANG ET AL.

1997; Widenfalk et aI., 1997; Basile and Hammerman, 1998). We, and others, have previously reported that these trophic factors are also neuroprotective. GDNF, given on the day of MCA ligation, or OP-l, given 24

hours before ischemia, decreases ischemiaireperfusion­induced brain infarction (Lin et aI., 1999; Wang et aI., 1997). The decrease in cortical infarction and restoration of behavioral deficits after fetal kidney grafting could represent a summation or interaction of several of these trophic factors. Moreover, previous studies have indi­cated that GDNF-induced responses are potentiated by OP-l (Bengtsson et aI., 1998) or TGF-13 (Krieglstein et aI., 1998). It is thus possible that fetal kidney transplants may provide a natural "cocktail" of various trophic fac­tors, which synergistically interact. Future experiments combining fetal kidney grafts and intracerebral injection of blocking antibodies will be needed to dissect out the contribution of the various TGF-13 family members (Messer et aI., 1998).

Previous studies have indicated that proteins in the TGF-13 superfamily are activated during degeneration and regeneration in adult tissues. The expression of TGF-131 mRNA is increased in regenerating renal tubules after acute ischemic injury (Basile et aI., 1996). Similarly, TGF-131 transcript expression is enhanced in hippocam­pus after transient forebrain ischemia (Klempt et aI., 1992). In vivo and in vitro studies have demonstrated that TGF-131 reduces global ischemia- or nitric oxide-induced damage in hippocampal CAl neurons (Henrich-Noack et aI., 1996). GDNF, a trophic factor of TGF-13 superfam­ily, has been shown to protect dopaminergic neurons from damage induced by neurotoxins that elevate intra­cellular free radicals and produce damage to mitochon­drial respiratory enzymes (Kearns and Gash, 1995;

Cheng et aI., 1998). We and others have reported that ligation of the MCA or local application of excitatory amino acids induces nitric oxide release from the cortical cells (Lin et aI., 1996; Liu et aI., 1997). Pretreatment with GDNF protects against both the cortical infarction and the elevation in nitric oxide formation (Wang et aI., 1997). Recent studies have further shown that GDNF pretreatment markedly reduces TUNEL labeling (Abe et aI., 1997) and caspase-l and 3 immunoreactivity (Kitagawa et aI., 1998a) in cortex during stroke. Taken together, these data suggest that TGF-13 superfamily mol­ecules may have neuroprotective effects mediated through the inhibition of apoptotic mechanisms and free radical production.

In conclusion, our data indicate that fetal kidney trans­plants have protective effects in the central nervous sys­tem and can reduce ischemia-induced injury in the adult cerebral cortex. The precise mechanisms for this action and determination of the involvement of specific trophic factors require further study. However, fetal kidney may

J Cereb Blood Flow Metab, Vol. 19, No. 12, 1999

act as a unique cellular reservoir for administration of multiple trophic factors to the injured brains.

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J Cereb Blood Flow Metab. Vol. 19. No. 12. 1999