- Email: [email protected]
Neural transplants disrupt the blood-brain barrier and allow peripherally acting drugs to exert a centrally mediated behavioral effect
102,1&b152 (1988)
EXPERIMENTALNEUROLOGY
BRIEF COMMUNICATION Neural Transplants Disrupt the Blood-Brain Barrier and Allow Peripherally Acting Drugs to Exert a Centrally Mediated Behavioral Effect PAULR.SANBERG,DAVIDR.NASH,STEPHENF.CALDERON,MAGDAGIORDANO, MICHAEL T. SHIPLEY,ANDANDREW B-NORMAN Division
of Neuroscience, University
Departments of Cincinnati
of Psychiatry, Neurosurgery, Physiology, Psychology, College of Medicine, Cincinnati, Ohio 45267-0559
The disruption of the blood-brain barrier (BBB) following neural transplantation has been demonstrated with horseradish peroxidase histochemistry. It appears that the BBB becomes at least temporarily permeable to large macromolecules. In this study, two drugs (Nmethylscopolamine and domperidone) that do not normally cross the BBB were shown to exert a centrally mediated behavioral effect when systematically administered in transplanted rats. This demonstrates that Nmethylscopolamine, domperidone, and perhaps other peripherally acting drugs can enter the brain via transplants and directly modify CNS function. o 1~88 Academic Press,
Inc.
Neural tissue transplants have enormous potential as a method of treatment for many CNS disorders. Recently, however, it has been reported that the bloodbrain barrier (BBB) does not develop normally within transplanted tissue (16-18). The BBB plays an important role in maintaining homeostasis within the brain, and its disruption may have a definite impact on the usefulness of brain tissue transplants. Large molecules, such as horseradish peroxidase (HRP 40 kDa), which do not normally enter the brain following systemic injection can be found within transplanted tissue, surrounding host tissue, and in the cerebrospinal fluid (CSF). In this study, we examined the status of the BBB from a new perspective using behavioral techniques, in addition to the previously used HRP histocytochemistry method. It was found that two drugs that do not normally pass the BBB had centrally mediated behavioral effects when systemically injected in transplanted animals. N-Methylscopolamine and domperidone both were able to influence behavior when a route of entry was made available vis-a-vis striatal transplants into the brain, suggesting the possibility that transplants might serve as a site of access for chemotherapeutic agents in the brain. Domperidone, a Dz dopamine receptor antagonist (lo), and N-methylscopolamine, a muscarinic receptor antagonist (8), were utilized because the behavioral effects of drugs with similar receptor binding character-
and Anatomy,
istics that do cross the BBB have been well studied. In normal animals, a Dz dopamine receptor antagonist that crossesthe BBB would be expected to inhibit the stereotypy induced by the dopamine agonist apomorphine. Therefore, it was determined whether systemic injections of domperidone which does not normally cross the BBB were able to inhibit apomorphine-induced stereotypy following neural transplants. Scopolamine, the nonmethylated form of N-methylscopolamine, does cross the BBB in normal animals and interacts with the striatal cholinergic system to elicit specific behavioral effects, such as increased locomotor behavior (8, 9, 22, 23). Thus, we determined whether N-methylscopolamine caused scopolamine-like behavioral effects in animals that had neural transplants. Two experiments were designed: one for domperidone and the other for N-methylscopolamine. In both experiments adult male Sprague-Dawley rats received bilateral microinjections of solid grafts (2 mm3 per side) of either Days 17-19 fetal striatal tissue or Ringer’s solution (sham transplant group) into unlesioned striatum (1, 5, 7, 14). In the domperidone experiment, 5 weeks after surgery the rats were divided into two groups and given injections of either domperidone (1 mg/kg ip) or saline. Thirty minutes later the rats were challenged with apomorphine (0.5 mg/kg SC) and visually assessed for stereotypy (4, 12). In the N-methylscopolamine experiment, 3 weeks after surgery the animals were placed into automated Digiscan Animal Activity monitors (Omnitech Electronics, Inc.) and habituated for 1 h (21). They were then injected with either N-methylscopolamine (2,4, or 8, mg/kg ip) or saline and their locomotor behavior was tested for 1 h. Three days later the drug treatment was reversed in both the domperidone and the N-methylscopolamine experiments. The integrity of the BBB was studied using horseradish peroxidase histochemistry 6 weeks after transplant. Animals were anesthetized and injected into the left cardiac ventricle with 1 ml of 5% HRP which was allowed to circulate for 2 min after which the animals were immediately perfused with normal saline for 5 min followed by 1% paraformaldehyde, 1.25% glutaraldehyde, and 2%
149 All
Copyright 0 1988 rights of reproduction
0014-4886/68 $3.00 by Academic Press, Inc. in any form reserved.
150
SANBERG SALINE
& APOMORPHINE
(0.5mg/kg)
5
:ONTROL
DOMPERIDONE
I1
(1 mg/kg)
CONTROL
I
-\A TRANSPLANT
6( APOMORHINE(0.5mg/kg)
TRANSPLANT
FIG. 1. The effect of domperidone pretreatment on stereotypy in rats with striatal transplants and controls with sham transplants. Rats received bilateral stereotaxic injections of either solid fetal striatal tissue (2 mm3 per side) or the same volume of vehicle. Four to six weeks later rats were placed in an open-field environment and left to habituate for 30 min. Rats were then injected with domperidone (1 mg/kp ip) or saline. Thirty minutes later all rats were injected with apomorphine (0.5 mg/kg SC) and visually rated for stereotypy for 1 min every 10 min over a 50-min session. Three to five days later the groups were reversed and reassessed for stereotypy. Stereotypy values represent the mean values f SE from groups of eight rats. Asterisk denotes statistical significance, P < 0.05 (Student’s t test).
ET
AL.
polamine produced a statistically significant increase in center time at all doses in animals with fetal striatal transplants; the effect on sham transplants was not significantly different from saline-treated controls (see Table 1). Examination of the data indicated that N-methylscopolamine produced an inverted U-shaped dose-response curve for center time in the rats with fetal striatal transplants. This is similar to the action of scopolamine which does cross the BBB and also produces an inverted U-shaped dose-response curve for center time in normal animals in this same paradigm (22). Animals with fetal striatal transplants showed a significant increase in vertical activity at one dose (4 mg/kg), while the other doses and the sham transplanted animals showed no significant change in vertical activity from saline-treated controls. The vertical activity variable displayed an interesting dose-response curve in fetal striatal transplanted animals: the middle dose produced an effect but neither the high dose (8 mg/kg) nor the low dose (2 mg/kg) produced a significant effect. It is difficult to find a correlation with this pattern and the effect of scopolamine on normal animals. The effect of scopolamine on vertical activity in normal animals is complex and not well studied, but it appears to depend on dose, previous exposure to the drug, and the size of the testing apparatus (9). The fact that domperidone pretreatment was able to antagonize apomorphine-induced stereotypy indicated that it has a centrally mediated pharmacological effect in fetal striatal transplanted animals. Since domperidone had no effect in sham transplanted animals, it is likely that the fetal transplant modified the permeability of the BBB for at least 5 weeks. The results with Nmethylscopolamine also suggested that the BBB remains disrupted after fetal striatal transplantation for
TABLE
sucrose all in 0.1 M phosphate buffer; 30 min later the perfusate was changed to 2% sucrose in 0.1 Mphosphate buffer for 30 min to clear fixative. The brains were removed and stored overnight in 20% sucrose in 0.1 M phosphate buffer; frozen sections (50 &f) were collected and processed for histochemistry with tetramethylbenzidine (TMB) (13,25). Pretreatment with domperidone significantly decreased the amount of apomorphine-induced stereotypy in animals with fetal striatal transplants (see Fig. 1). In contrast, neither domperidone nor saline pretreatment had any effect on the apomorphine-induced stereotypy in animals with sham transplants. The results suggest that in the transplanted animals the domperidone crossed the BBB and antagonized the effects of apomorphine. The effect of N-methylscopolamine on animals with fetal striatal and sham transplants was evaluated in terms of center time and vertical activity as a percentage of the value produced by saline treatment. N-Methylsco-
1
The Effect of N-Methylscopolamine on Digiscan Activity Variables of Center Time and Vertical Activity in Bats with Fetal Striatal or Sham Transplants Diiscan variable Center
Vertical
time
activity
Dose bdk)
Striatal transplants
(X)
Sham transplant
2 4 8
274* 530* 234*
56 60 137
2 4 8
81 456* 157
111 162 145
(%)
Note. Rats received either bilateral fetal striatal or sham transplants. Two to four weeks later rats were placed in Digiscan activity monitors and left to habituate for 60 min. Rats were then injected with either I’+-methylscopolamine (2,4,8 mg/kg ip) or saline and their locomotor behavior was tested for 1 h. Three days later the groups were reversed and reassessed for locomotor activity. Data represent the N-methylscopolamine results as a percentage of saline values for each group of animals (n = I/group). Asterisk denotes statistically significant change in activity, P < 0.05.
BLOOD-BRAIN
BARRIER
FIG. 2. Bilateral fetal striatal grafts (17-19 days) were transplanted performed 6 weeks after transplants. The HRP was allowed to circulate histology with tetramethylbenzidine (TMB). The area of the transplant of extension into the surrounding tissue. A rim of reactive nroduct is vessels in the meninges. Tr, transplant site; S, striatum.
at least 3 weeks. Surgical trauma, per se, did not appear to alter the BBB as there was no effect with either domperidone or N-methylscopolamine in animals receiving injections of Ringer’s solution (6,28). Recently, Wakai et al. (27) using autogenic skin, muscle, and allogenic superior cervical ganglia (SCG) and Rosenstein using allogenic fetal neocortex, superior cervical ganglia, and adrenal medulla (16-19) have demonstrated that the transplant leaks HRP, especially within the first few months. Our HRP results are consistent with these findings. We found heavy HRP infiltration in the area of the striatal transplant 6 weeks after surgery and virtually no HRP in those animals receiving sham transplants (see Fig. 2). The fact that vessels are permeable to HRP 6 weeks after surgery is consistent with both N-methylscopolamine and domperidone exerting a centrally mediated effect during this period. Interestingly, Wakai et al. (27) and Rosenstein and Phillips (20) have reported that the BBB recovers in transplants. Wakai et al. (27) report recovery 6-12 months following transplant, and Rosenstein and Phillips (20) report recovery by 3 months following intraparenchymal transplant. Similarly, other investigators have reported an intact BBB in a variety of other transplant models (3,11, 26, 29). Preliminary studies in our model indicate that HRP does not significantly penetrate brain parenchyma 6 months after surgery (unpublished observations). The intraparenchymal transplant appears to disrupt the BBB significantly longer than either an injection of vehicle
AND
TRANSPLANTS
151
into the unlesioned adult striatum. Histocytochemistry using HRP was 2 min and then the tissue was fixed. Collected tissue was processed for is completely filled with reaction product along with a moderate amount also present surrounding the entire brain possibly representing leaky
solution or injury by a penetrating lesion (6, 18, 27). The mechanism by which the transplant prolongs the period of BBB disruption can only be hypothesized at this time. It is possible that the fetal transplanted tissue contains a growth factor or neurohumoral agent that promotes angiogenesis. It is well established that during angiogenesis vessels are permeable to macromolecules (15, 24). If animals receiving fetal tissue transplants do have prolonged angiogenesis, then this might explain the increase in vessel permeability of the transplanted animals. This is the first demonstration in which peripherally acting drugs enter the brain via transplant and directly modify CNS function. This suggests the intriguing possibility that transplants might serve as a site of access for allowing chemotherapeutic agents into the brain. Three aspects of transplant disruption of the BBB have potential clinical significance. First, transplants can be located at the exact site of desired drug delivery; therefore undesired nonspecific effects elsewhere in the brain may be minimized. Second, the BBB appears to recover in time, thereby minimizing long-term adverse effects of a leaky BBB. Finally, it may be possible to use trophic factors or other agents that normally do not cross the BBB to enhance the integration of the transplant with the host (2) since they would accumulate preferentially in the transplant. These clinical possibilities are interesting and merit further research into the potential of neural transplants to target drug therapy delivery into specific brain regions.
152
SANBERG ACKNOWLEDGMENTS
This research was supported by grants from the Pratt Family and Friends, the Hereditary Disease Foundation, the Huntington’s Disease Foundation of America, the National Institutes of Health (ROl NS25647), the URC, the U.S. Army, an Alzheimer’s Research Center grant from the Ohio Department of Aging, and Omnitech Electronics, Inc. (Columbus, OH), to P. R. Sanberg, M. T. Shipley, and A. B. Norman. We also gratefully acknowledge the assistance of R. Durbin and T. Burkhart. Note added in proof. Recently, Broadwell and Rosenstein have exchanged dialogue on whether a blood-brain barrier does or does not develop in neural transplants. Readers are referred to Science 241: 473-474,1988.
REFERENCES 1. BJ~~RKLUND, A., V. STENEVI, R. H. SCHMIDT, S. B. DUNNETT, AND F. H. GAGE. 1983. Introduction and general methods of preparation for intracerebral grafting of neuronal cell suspensions. Acta Physiol. &and. 522(Suppl.): l-7. 2. BJBRKLUND, A., 0. LINDVALL, 0. ISACSON, P. BRUNDIN, K. WICTORIN, R. E. STRECKER, D. J. CLARKE, AND S. B. DUNNETT. 1987. Mechanism of action of intracerebral neural transplants: Studies on nigral and striatal grafts to the lesioned striatum. Trends Neurosci. 10: 509-516. 3. BROADWELL, R., M. CHARLTON, M. SLACMAN, AND R. SCHLEGEL. 1987. Allografts of CNS tissue do exhibit a blood-brain barrier to protein. Sot. Neurosci. Abstr. 13: 1250. 4. CREESE, I., AND S. D. IVERSON. 1973. Blockade of amphetamineinduced motor stimulation and sterotypy in the adult rat following neonatal treatment with 6-hydroxydopamine. Brain Res. 56: 369-382.
5. DAS, G. D. 1983. Neural transplantation in mammalian brain: Some conceptual and technical considerations. Pages l-64 in R. B. WALLACE AND G. D. DAS, Eds., Neural Tissue Transplantation Research. Springer-Verlag, New York. 6. FARRELL, J. B., G. S. SARNA, AND M. E. CARNEY. 1987. Bloodbrain barrier and restitution following brain injury. Sot. Neurosci. Abstr.
13: 774.
7. HAGENMEYER-HOUSER, S., AND P. R. SANBERG. 1987. Locomotor behavior changes induced by E-17 striatal transplants in normal rats. Pharmacol. Biochem. Behav. 27: 583-586. 8. HUGHES, R. A. 1982. Anticholinergic drugs, blood-brain barrier, and tonic immobility in chickens. Physiol. Behav. 29: 67-71. 9. HUGHES, R. N. 1982. A review of atropinic drug effects on exploratory choice behavior in laboratory rodents. Behav. Neural Biol. 34: 5-41. 10. LADURON, P. M., AND J. E. LEYSEN. 1979. Domperidone, a specific in vitro dopamine antagonist, devoid of in vivo central dopaminergic activity. Bioehem. Pharmacol. 28: 2161-2165. 11. LEE, M. H., A. S. LOSSINSKY, A. RABE, AND H. M. WISNIEWSKI. 1987. Intact blood-brain barrier in large neocortical transplants in the rat brain. Sot. Neurosci. Abstr. 13: 289.
ET AL. 12. MASON, S. T., P. R. SANBERG, AND H. D. FIBIGER. 1978. Kainic acid lesions of the striatum dissociate amphetamine and apomorphine stereotypy: Similarities to Huntington’s chorea. Science 201: 352-355. 13 for horseradish ’ MESULAM, M. M. 1978. Tetramethylbenzidine peroxidase neurohistochemistry: A non-carcinogenic blue reaction with superior sensitivity for visualizing neural afferents and efferents. J. Histochem. Cytochem. 26: 106-117. PAXINOS, G., AND C. WATSON. 1984. The Rat Brain in Stereotaxic 14’ Coordinates. Academic Press, Sydney/San Diego. 15. ROBBINS, S. L., R. S. COTRAN, AND V. KUMAR. 1984. Pathologic Basis of Disease. Saunders, Philadelphia. 16. ROSENSTEIN, J. M. 1987. Neocortical transplants in the mammalian brain lack a blood-brain barrier to macromolecules. Science 235: 772-774. 17. ROSENSTEIN, J. M., AND M. W. BRIGHTMAN. 1983. Circumventing the BBB with autonomic ganglion transplants. Science 221: 879-881. 18. ROSENSTEIN, J. M. 1987. Adrenal medulla grafts produce bloodbrain barrier dysfunction. Brain Res. 414: 192-196. 19. ROSENSTEIN, J. M., AND M. W. BRIGHTMAN. 1986. Alterations of the blood-brain barrier after transplantation of autonomic ganglia into the mammalian central nervous system. J. Comp. Neural. 250: 339-351. 20. ROSENSTEIN, J. M., AND T. M. PHILLIPS. 1988. Blood brain and blood cerebral spinal fluid alterations following neural transplants. Prog. Brain Res., in press. 21. SANBERG, P. R., S. H. HAGENMEYER, AND M. A. HENAULT. 1985. Automated measurements of multivariate locomotor behavior in rodents. Neurobehav. Toxicol. Teratol. 7: 87-94. 22. SANBERG, P. R., S. A. ZOLOTY, R. WILLIS, C. D. TICARICH, K. RHOADS, R. P. NAGY, S. G. MITCHELL, A. R. LAFOREST, J. A. JENKS, L. J. HARKABUS, D. B. GURSON, J. A. FINNEFROCK, AND E. J. BEDNARIK. 1987. Dig&can activity: Automated measurement of thigmotactic and stereotypic behavior in rats. Pharmacol. Biochem.
Behav.
27:
569-572.
23. SANBERG, P. R., M. A. HENAULT, S. H. HAGENMEYER-HOUSER, AND K. H. RUSSELL. 1987. The topography of amphetamine- and scopolamine-induced hyperactivity: Toward an activity print. Beh.av. Neurosci. 101: 131-133. 24. SCHOEFL, G. I. 1963. Studies on inflamation. III. Growing capillaries: Their structure and permeability. Virchows Arch. [Pathol. Anat.] 337: 97-141. 25. SHIPLEY, M. T. 1982. Insular cortex projection to the nucleus of the solitary tract and brainstem viscoromotor regions in the mouse. Brain Res. Bull. 3: 139-148. 26. SWENSON, R. S., J. C. SORENSEN, J. ZIMMER, AND A. J. CASTRO. 1987. Neocortical transplants grafted into the newborn rat brain demonstrate a blood-brain barrier to macromolecules. Sot. Neurosci. Abstr. 13: 289. 27. WAKAI, S., S. E. MEISELMAN, AND M. W. BRIGHTMAN. 1986. Focal circumvention of blood-brain barrier with grafts of muscle, skin, and autonomic ganglion. Brain Res. 386: 209-222. 28. WAKAI, S., S. E. MEISELMAN, AND M. W. BFUGHTMAN. 1986. Muscle grafts vs. entries for blood-borne proteins into the extracellular space of the brain. Neurosurgery 18: 548-554. 29. WELLS, J., B. P. VIETGE, D. G. WELLS, AND M. FIALA. 1987. Repair of the blood-brain barrier during the first week after septohippocampal transplants. Sot. Neurosci. Abstr. 13: 287.
EXPERIMENTALNEUROLOGY
BRIEF COMMUNICATION Neural Transplants Disrupt the Blood-Brain Barrier and Allow Peripherally Acting Drugs to Exert a Centrally Mediated Behavioral Effect PAULR.SANBERG,DAVIDR.NASH,STEPHENF.CALDERON,MAGDAGIORDANO, MICHAEL T. SHIPLEY,ANDANDREW B-NORMAN Division
of Neuroscience, University
Departments of Cincinnati
of Psychiatry, Neurosurgery, Physiology, Psychology, College of Medicine, Cincinnati, Ohio 45267-0559
The disruption of the blood-brain barrier (BBB) following neural transplantation has been demonstrated with horseradish peroxidase histochemistry. It appears that the BBB becomes at least temporarily permeable to large macromolecules. In this study, two drugs (Nmethylscopolamine and domperidone) that do not normally cross the BBB were shown to exert a centrally mediated behavioral effect when systematically administered in transplanted rats. This demonstrates that Nmethylscopolamine, domperidone, and perhaps other peripherally acting drugs can enter the brain via transplants and directly modify CNS function. o 1~88 Academic Press,
Inc.
Neural tissue transplants have enormous potential as a method of treatment for many CNS disorders. Recently, however, it has been reported that the bloodbrain barrier (BBB) does not develop normally within transplanted tissue (16-18). The BBB plays an important role in maintaining homeostasis within the brain, and its disruption may have a definite impact on the usefulness of brain tissue transplants. Large molecules, such as horseradish peroxidase (HRP 40 kDa), which do not normally enter the brain following systemic injection can be found within transplanted tissue, surrounding host tissue, and in the cerebrospinal fluid (CSF). In this study, we examined the status of the BBB from a new perspective using behavioral techniques, in addition to the previously used HRP histocytochemistry method. It was found that two drugs that do not normally pass the BBB had centrally mediated behavioral effects when systemically injected in transplanted animals. N-Methylscopolamine and domperidone both were able to influence behavior when a route of entry was made available vis-a-vis striatal transplants into the brain, suggesting the possibility that transplants might serve as a site of access for chemotherapeutic agents in the brain. Domperidone, a Dz dopamine receptor antagonist (lo), and N-methylscopolamine, a muscarinic receptor antagonist (8), were utilized because the behavioral effects of drugs with similar receptor binding character-
and Anatomy,
istics that do cross the BBB have been well studied. In normal animals, a Dz dopamine receptor antagonist that crossesthe BBB would be expected to inhibit the stereotypy induced by the dopamine agonist apomorphine. Therefore, it was determined whether systemic injections of domperidone which does not normally cross the BBB were able to inhibit apomorphine-induced stereotypy following neural transplants. Scopolamine, the nonmethylated form of N-methylscopolamine, does cross the BBB in normal animals and interacts with the striatal cholinergic system to elicit specific behavioral effects, such as increased locomotor behavior (8, 9, 22, 23). Thus, we determined whether N-methylscopolamine caused scopolamine-like behavioral effects in animals that had neural transplants. Two experiments were designed: one for domperidone and the other for N-methylscopolamine. In both experiments adult male Sprague-Dawley rats received bilateral microinjections of solid grafts (2 mm3 per side) of either Days 17-19 fetal striatal tissue or Ringer’s solution (sham transplant group) into unlesioned striatum (1, 5, 7, 14). In the domperidone experiment, 5 weeks after surgery the rats were divided into two groups and given injections of either domperidone (1 mg/kg ip) or saline. Thirty minutes later the rats were challenged with apomorphine (0.5 mg/kg SC) and visually assessed for stereotypy (4, 12). In the N-methylscopolamine experiment, 3 weeks after surgery the animals were placed into automated Digiscan Animal Activity monitors (Omnitech Electronics, Inc.) and habituated for 1 h (21). They were then injected with either N-methylscopolamine (2,4, or 8, mg/kg ip) or saline and their locomotor behavior was tested for 1 h. Three days later the drug treatment was reversed in both the domperidone and the N-methylscopolamine experiments. The integrity of the BBB was studied using horseradish peroxidase histochemistry 6 weeks after transplant. Animals were anesthetized and injected into the left cardiac ventricle with 1 ml of 5% HRP which was allowed to circulate for 2 min after which the animals were immediately perfused with normal saline for 5 min followed by 1% paraformaldehyde, 1.25% glutaraldehyde, and 2%
149 All
Copyright 0 1988 rights of reproduction
0014-4886/68 $3.00 by Academic Press, Inc. in any form reserved.
150
SANBERG SALINE
& APOMORPHINE
(0.5mg/kg)
5
:ONTROL
DOMPERIDONE
I1
(1 mg/kg)
CONTROL
I
-\A TRANSPLANT
6( APOMORHINE(0.5mg/kg)
TRANSPLANT
FIG. 1. The effect of domperidone pretreatment on stereotypy in rats with striatal transplants and controls with sham transplants. Rats received bilateral stereotaxic injections of either solid fetal striatal tissue (2 mm3 per side) or the same volume of vehicle. Four to six weeks later rats were placed in an open-field environment and left to habituate for 30 min. Rats were then injected with domperidone (1 mg/kp ip) or saline. Thirty minutes later all rats were injected with apomorphine (0.5 mg/kg SC) and visually rated for stereotypy for 1 min every 10 min over a 50-min session. Three to five days later the groups were reversed and reassessed for stereotypy. Stereotypy values represent the mean values f SE from groups of eight rats. Asterisk denotes statistical significance, P < 0.05 (Student’s t test).
ET
AL.
polamine produced a statistically significant increase in center time at all doses in animals with fetal striatal transplants; the effect on sham transplants was not significantly different from saline-treated controls (see Table 1). Examination of the data indicated that N-methylscopolamine produced an inverted U-shaped dose-response curve for center time in the rats with fetal striatal transplants. This is similar to the action of scopolamine which does cross the BBB and also produces an inverted U-shaped dose-response curve for center time in normal animals in this same paradigm (22). Animals with fetal striatal transplants showed a significant increase in vertical activity at one dose (4 mg/kg), while the other doses and the sham transplanted animals showed no significant change in vertical activity from saline-treated controls. The vertical activity variable displayed an interesting dose-response curve in fetal striatal transplanted animals: the middle dose produced an effect but neither the high dose (8 mg/kg) nor the low dose (2 mg/kg) produced a significant effect. It is difficult to find a correlation with this pattern and the effect of scopolamine on normal animals. The effect of scopolamine on vertical activity in normal animals is complex and not well studied, but it appears to depend on dose, previous exposure to the drug, and the size of the testing apparatus (9). The fact that domperidone pretreatment was able to antagonize apomorphine-induced stereotypy indicated that it has a centrally mediated pharmacological effect in fetal striatal transplanted animals. Since domperidone had no effect in sham transplanted animals, it is likely that the fetal transplant modified the permeability of the BBB for at least 5 weeks. The results with Nmethylscopolamine also suggested that the BBB remains disrupted after fetal striatal transplantation for
TABLE
sucrose all in 0.1 M phosphate buffer; 30 min later the perfusate was changed to 2% sucrose in 0.1 Mphosphate buffer for 30 min to clear fixative. The brains were removed and stored overnight in 20% sucrose in 0.1 M phosphate buffer; frozen sections (50 &f) were collected and processed for histochemistry with tetramethylbenzidine (TMB) (13,25). Pretreatment with domperidone significantly decreased the amount of apomorphine-induced stereotypy in animals with fetal striatal transplants (see Fig. 1). In contrast, neither domperidone nor saline pretreatment had any effect on the apomorphine-induced stereotypy in animals with sham transplants. The results suggest that in the transplanted animals the domperidone crossed the BBB and antagonized the effects of apomorphine. The effect of N-methylscopolamine on animals with fetal striatal and sham transplants was evaluated in terms of center time and vertical activity as a percentage of the value produced by saline treatment. N-Methylsco-
1
The Effect of N-Methylscopolamine on Digiscan Activity Variables of Center Time and Vertical Activity in Bats with Fetal Striatal or Sham Transplants Diiscan variable Center
Vertical
time
activity
Dose bdk)
Striatal transplants
(X)
Sham transplant
2 4 8
274* 530* 234*
56 60 137
2 4 8
81 456* 157
111 162 145
(%)
Note. Rats received either bilateral fetal striatal or sham transplants. Two to four weeks later rats were placed in Digiscan activity monitors and left to habituate for 60 min. Rats were then injected with either I’+-methylscopolamine (2,4,8 mg/kg ip) or saline and their locomotor behavior was tested for 1 h. Three days later the groups were reversed and reassessed for locomotor activity. Data represent the N-methylscopolamine results as a percentage of saline values for each group of animals (n = I/group). Asterisk denotes statistically significant change in activity, P < 0.05.
BLOOD-BRAIN
BARRIER
FIG. 2. Bilateral fetal striatal grafts (17-19 days) were transplanted performed 6 weeks after transplants. The HRP was allowed to circulate histology with tetramethylbenzidine (TMB). The area of the transplant of extension into the surrounding tissue. A rim of reactive nroduct is vessels in the meninges. Tr, transplant site; S, striatum.
at least 3 weeks. Surgical trauma, per se, did not appear to alter the BBB as there was no effect with either domperidone or N-methylscopolamine in animals receiving injections of Ringer’s solution (6,28). Recently, Wakai et al. (27) using autogenic skin, muscle, and allogenic superior cervical ganglia (SCG) and Rosenstein using allogenic fetal neocortex, superior cervical ganglia, and adrenal medulla (16-19) have demonstrated that the transplant leaks HRP, especially within the first few months. Our HRP results are consistent with these findings. We found heavy HRP infiltration in the area of the striatal transplant 6 weeks after surgery and virtually no HRP in those animals receiving sham transplants (see Fig. 2). The fact that vessels are permeable to HRP 6 weeks after surgery is consistent with both N-methylscopolamine and domperidone exerting a centrally mediated effect during this period. Interestingly, Wakai et al. (27) and Rosenstein and Phillips (20) have reported that the BBB recovers in transplants. Wakai et al. (27) report recovery 6-12 months following transplant, and Rosenstein and Phillips (20) report recovery by 3 months following intraparenchymal transplant. Similarly, other investigators have reported an intact BBB in a variety of other transplant models (3,11, 26, 29). Preliminary studies in our model indicate that HRP does not significantly penetrate brain parenchyma 6 months after surgery (unpublished observations). The intraparenchymal transplant appears to disrupt the BBB significantly longer than either an injection of vehicle
AND
TRANSPLANTS
151
into the unlesioned adult striatum. Histocytochemistry using HRP was 2 min and then the tissue was fixed. Collected tissue was processed for is completely filled with reaction product along with a moderate amount also present surrounding the entire brain possibly representing leaky
solution or injury by a penetrating lesion (6, 18, 27). The mechanism by which the transplant prolongs the period of BBB disruption can only be hypothesized at this time. It is possible that the fetal transplanted tissue contains a growth factor or neurohumoral agent that promotes angiogenesis. It is well established that during angiogenesis vessels are permeable to macromolecules (15, 24). If animals receiving fetal tissue transplants do have prolonged angiogenesis, then this might explain the increase in vessel permeability of the transplanted animals. This is the first demonstration in which peripherally acting drugs enter the brain via transplant and directly modify CNS function. This suggests the intriguing possibility that transplants might serve as a site of access for allowing chemotherapeutic agents into the brain. Three aspects of transplant disruption of the BBB have potential clinical significance. First, transplants can be located at the exact site of desired drug delivery; therefore undesired nonspecific effects elsewhere in the brain may be minimized. Second, the BBB appears to recover in time, thereby minimizing long-term adverse effects of a leaky BBB. Finally, it may be possible to use trophic factors or other agents that normally do not cross the BBB to enhance the integration of the transplant with the host (2) since they would accumulate preferentially in the transplant. These clinical possibilities are interesting and merit further research into the potential of neural transplants to target drug therapy delivery into specific brain regions.
152
SANBERG ACKNOWLEDGMENTS
This research was supported by grants from the Pratt Family and Friends, the Hereditary Disease Foundation, the Huntington’s Disease Foundation of America, the National Institutes of Health (ROl NS25647), the URC, the U.S. Army, an Alzheimer’s Research Center grant from the Ohio Department of Aging, and Omnitech Electronics, Inc. (Columbus, OH), to P. R. Sanberg, M. T. Shipley, and A. B. Norman. We also gratefully acknowledge the assistance of R. Durbin and T. Burkhart. Note added in proof. Recently, Broadwell and Rosenstein have exchanged dialogue on whether a blood-brain barrier does or does not develop in neural transplants. Readers are referred to Science 241: 473-474,1988.
REFERENCES 1. BJ~~RKLUND, A., V. STENEVI, R. H. SCHMIDT, S. B. DUNNETT, AND F. H. GAGE. 1983. Introduction and general methods of preparation for intracerebral grafting of neuronal cell suspensions. Acta Physiol. &and. 522(Suppl.): l-7. 2. BJBRKLUND, A., 0. LINDVALL, 0. ISACSON, P. BRUNDIN, K. WICTORIN, R. E. STRECKER, D. J. CLARKE, AND S. B. DUNNETT. 1987. Mechanism of action of intracerebral neural transplants: Studies on nigral and striatal grafts to the lesioned striatum. Trends Neurosci. 10: 509-516. 3. BROADWELL, R., M. CHARLTON, M. SLACMAN, AND R. SCHLEGEL. 1987. Allografts of CNS tissue do exhibit a blood-brain barrier to protein. Sot. Neurosci. Abstr. 13: 1250. 4. CREESE, I., AND S. D. IVERSON. 1973. Blockade of amphetamineinduced motor stimulation and sterotypy in the adult rat following neonatal treatment with 6-hydroxydopamine. Brain Res. 56: 369-382.
5. DAS, G. D. 1983. Neural transplantation in mammalian brain: Some conceptual and technical considerations. Pages l-64 in R. B. WALLACE AND G. D. DAS, Eds., Neural Tissue Transplantation Research. Springer-Verlag, New York. 6. FARRELL, J. B., G. S. SARNA, AND M. E. CARNEY. 1987. Bloodbrain barrier and restitution following brain injury. Sot. Neurosci. Abstr.
13: 774.
7. HAGENMEYER-HOUSER, S., AND P. R. SANBERG. 1987. Locomotor behavior changes induced by E-17 striatal transplants in normal rats. Pharmacol. Biochem. Behav. 27: 583-586. 8. HUGHES, R. A. 1982. Anticholinergic drugs, blood-brain barrier, and tonic immobility in chickens. Physiol. Behav. 29: 67-71. 9. HUGHES, R. N. 1982. A review of atropinic drug effects on exploratory choice behavior in laboratory rodents. Behav. Neural Biol. 34: 5-41. 10. LADURON, P. M., AND J. E. LEYSEN. 1979. Domperidone, a specific in vitro dopamine antagonist, devoid of in vivo central dopaminergic activity. Bioehem. Pharmacol. 28: 2161-2165. 11. LEE, M. H., A. S. LOSSINSKY, A. RABE, AND H. M. WISNIEWSKI. 1987. Intact blood-brain barrier in large neocortical transplants in the rat brain. Sot. Neurosci. Abstr. 13: 289.
ET AL. 12. MASON, S. T., P. R. SANBERG, AND H. D. FIBIGER. 1978. Kainic acid lesions of the striatum dissociate amphetamine and apomorphine stereotypy: Similarities to Huntington’s chorea. Science 201: 352-355. 13 for horseradish ’ MESULAM, M. M. 1978. Tetramethylbenzidine peroxidase neurohistochemistry: A non-carcinogenic blue reaction with superior sensitivity for visualizing neural afferents and efferents. J. Histochem. Cytochem. 26: 106-117. PAXINOS, G., AND C. WATSON. 1984. The Rat Brain in Stereotaxic 14’ Coordinates. Academic Press, Sydney/San Diego. 15. ROBBINS, S. L., R. S. COTRAN, AND V. KUMAR. 1984. Pathologic Basis of Disease. Saunders, Philadelphia. 16. ROSENSTEIN, J. M. 1987. Neocortical transplants in the mammalian brain lack a blood-brain barrier to macromolecules. Science 235: 772-774. 17. ROSENSTEIN, J. M., AND M. W. BRIGHTMAN. 1983. Circumventing the BBB with autonomic ganglion transplants. Science 221: 879-881. 18. ROSENSTEIN, J. M. 1987. Adrenal medulla grafts produce bloodbrain barrier dysfunction. Brain Res. 414: 192-196. 19. ROSENSTEIN, J. M., AND M. W. BRIGHTMAN. 1986. Alterations of the blood-brain barrier after transplantation of autonomic ganglia into the mammalian central nervous system. J. Comp. Neural. 250: 339-351. 20. ROSENSTEIN, J. M., AND T. M. PHILLIPS. 1988. Blood brain and blood cerebral spinal fluid alterations following neural transplants. Prog. Brain Res., in press. 21. SANBERG, P. R., S. H. HAGENMEYER, AND M. A. HENAULT. 1985. Automated measurements of multivariate locomotor behavior in rodents. Neurobehav. Toxicol. Teratol. 7: 87-94. 22. SANBERG, P. R., S. A. ZOLOTY, R. WILLIS, C. D. TICARICH, K. RHOADS, R. P. NAGY, S. G. MITCHELL, A. R. LAFOREST, J. A. JENKS, L. J. HARKABUS, D. B. GURSON, J. A. FINNEFROCK, AND E. J. BEDNARIK. 1987. Dig&can activity: Automated measurement of thigmotactic and stereotypic behavior in rats. Pharmacol. Biochem.
Behav.
27:
569-572.
23. SANBERG, P. R., M. A. HENAULT, S. H. HAGENMEYER-HOUSER, AND K. H. RUSSELL. 1987. The topography of amphetamine- and scopolamine-induced hyperactivity: Toward an activity print. Beh.av. Neurosci. 101: 131-133. 24. SCHOEFL, G. I. 1963. Studies on inflamation. III. Growing capillaries: Their structure and permeability. Virchows Arch. [Pathol. Anat.] 337: 97-141. 25. SHIPLEY, M. T. 1982. Insular cortex projection to the nucleus of the solitary tract and brainstem viscoromotor regions in the mouse. Brain Res. Bull. 3: 139-148. 26. SWENSON, R. S., J. C. SORENSEN, J. ZIMMER, AND A. J. CASTRO. 1987. Neocortical transplants grafted into the newborn rat brain demonstrate a blood-brain barrier to macromolecules. Sot. Neurosci. Abstr. 13: 289. 27. WAKAI, S., S. E. MEISELMAN, AND M. W. BRIGHTMAN. 1986. Focal circumvention of blood-brain barrier with grafts of muscle, skin, and autonomic ganglion. Brain Res. 386: 209-222. 28. WAKAI, S., S. E. MEISELMAN, AND M. W. BFUGHTMAN. 1986. Muscle grafts vs. entries for blood-borne proteins into the extracellular space of the brain. Neurosurgery 18: 548-554. 29. WELLS, J., B. P. VIETGE, D. G. WELLS, AND M. FIALA. 1987. Repair of the blood-brain barrier during the first week after septohippocampal transplants. Sot. Neurosci. Abstr. 13: 287.