Turkish Neurosurgery 4: 21 - 26. 1994 Akalan: Blood-Brain Barrier Changes in Feta1 Transplants

Blood-Brain Barrier A1terations Foliowing Intracerebral Grafting:Solid and Celi Suspensions of Fetal Central Nervous System Tissue

NEJAT AKALAN. HAKAN SEÇKIN

Hacettepe University. School of Medidne. Department of Neurosurgery (NA) and Dr. Muhittin Ülker Adi Yardim ve TravmatolojiHospital. Clinic of Neurosurgery (HS). Ankara. Türkiye

Abstract : The time course of angiogenesis and the status of theBBBwithin solid and cell suspension grafts of feta! CNS tissuefollowing intracerebral transplantation is studied. Adult rats (n=40)received fetal forebrain tissue as either solid or cell suspensiongrafts to their corpus callosun bilaterally. The presence and theviability of the grafts was examined in fifty micron coronal sec­tions treated by cresyl violet (Nissi)and acetylcholinesterase (AChE)histochemistry. BBBpermeability within the grafts were determinedby horseradish peroxide (HRP)leakege given systemically on thefirst. third. seventh and tenth post-operative days. to four separate

INTRODUCTION

Neural transplantation is a significant tool to studythe aspects of development and plastidty in the cen­tral nervous system (CNS).as well as to demonstratethe ability of grafted neural tissue to restore certainphysiological dysfunction (UUO.29). Experimentalstudies with the extension to human subjects haveshown that the ho st and the graft but anatomicalevidence for such connections is limited. On the

other hand. the status of the blood-brain barrler (BBB)

following graft placement could have as yet undeter­mined consequences in terms of graft survival andamelioration of the functional disturbance. The bar­

rier changes after transplantation may serve to pro­mote graft survival by permitting transplanted cellsto be exposed to blood trophic substances. Mareaver,a disrupted BBB helps the ho st tissue to be exposedto the maaomol~cules and neurotransmitters within

the graft which in fact. is the aim of the transplanta­tion procedure. Therefore the status of BBB fol-

groups of rats (n:lO). HRP leakage was detected in both solid andcell suspension grafts on the first and third days followingtransplantation indicating a disrupted BBB. By the seventhpostoperative day. no HRP reaction was detected. These resultssuggest that the BBBpermeability of the vessels within intracerebralsolid grafts depends on the transplanted tissue itself while the hostCNS tissue provokes the formation of an intact BBBfor cell suspen­sion grafts.Key Words: Angiogenesis. Blood-brain barrier. Horseradishperoxide. Neura! transplantion.

lawing transplantation is of crucial importance andmanipulating the permeability of the vessels withinthe grafted tissue may provide a new therapeutic in­sight for human applications. On the other hand. ex­posure of the graft to host blood antibodies througha compromised BBB may provoke an immuneresponse to the grafted tissue resulting with graft re­jection. There is evidence that the duration of BBB

permeability correlates closely with the sequence ofimmunological rejection of the graft (23.35). In thepresent study the time course of angiogenesis andthe status of the BBB within solid and cell suspen­sian grafts of fetal CNS tissue following intracerebraltransplantation. are examined.

METHODS

Fetal tissue of CNS origin in either solid or cellsuspension form were transplanted into the cerebralparenchyma of adult male Sprague-Dawley ratsweighing approximately 250 grams. Fetal CNS tissue

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Turkish Neurosurgery 4: 21 - 26. 1994

was obtained from timed pregnat (15 and 16 dayspostcoitus) females. The donor rats were anesthetiz­ed with an intramuscular overdose of ketamine

hydrochloride (50 mg/ml) / xylazine hydrochloride(%2.5) solution and the fetuses were removed in­dividually under aseptic conditions. The basalforebrain tissue was dissected from the fetuses and

pooled in Hank's Buffered Saline Solution (HBSS).Pieces of tissue approximately 1.0 mm3 in size wereused as solid grafts. For the preparation of cellsuspension. the remaining pieces were gent1ytransferred to a microvial and 0.1% trypsin (Type II:crude. Sigma) in phosphate buffered saline (PBS)wasadded (300 microliters per dissected brain). Thesuspension was incubated at 37 COfor 20 minutes.The trypsin was removed and washed off three timeswith PBS. Finally 10 microliters pf the solution pertissue were added and the pieces that settled weredissociated into a suspension by repeated pipettingthrough a Pasteur pipette. The resulting milky fluidwas kept at room temperature un til the end of theexperiment. Ten microliters were injected stereotax­ically with aHarnilton syringe (26 nO.needle gauge)into the host corpus callosum of adult. male rats ofthe same strain. at coordinates 2.5 mm. anterior to

the bregma. 2.0 mm. off midline. 3.0 mm. deep fromthe cortical surface. The contralateral side received

a solid graft of fetal basal forebrain approximatelylxlxl mm. in size at the same coordinates. througha modified 20 gauge spinal needle. The basal foreb­rain region was specificaliy chosen because it is richin cholinergic neurons that cen be easily detected byacetyl cholinesterase (AChE) histochemistry. Thirtyanimals received both celi suspension and solid graftsof fetal CNS tissue. Recipients were kept at roomtemperature with water and food ad libidum.

The rats. divided into four group s (n:10 for each)were sacrified on the first. third. seventh and tenth

days after transplantation. respectively. Prior tosacrifice. each animal received diisopropylfuorop­hosphate (DBF).1.0 mg/kg/rat intramuscularly. to sup­press neurophil staining. Six hours following DBFin­jections. the animals were anesthetized andhorseradish peroxide (HRP)Type VI 0.75 mg/5 gmbody weight was given through the femoral vein.HRP was allowed to circulate for one bour and the

rats were perfused with 0.1 M PBS for 5 minutesfollowed by 1.25% glutaraldehyde / 1.0% parafor­maldehyde solution in PBS. Ten minutes later theperfusate was changed to PBS again to clear the fix-

22

Aka1an: BJood-Brain Barrier Changes in Feta1 TranspJi'lI1tS

ative. The brains were removed and stored overnightin the same fixative at 4 CO.Corona! sections of 50

microns through the forebrain were obtained by anOxford vibrotome. and submitted for aesyl violetstain. HRP and AchE histochemistry.

HRP histology was performed using thetetramethylbenzidine (TMB)method (20). HRP sec­tions were counterstained with neutral red prior toexamination. AChE histochemistry was performedaccording to the method of Naik (21).Serial sectionswere examined by AchE and aesyl violet stainingfor the presence and location of the graft and BBBleakage was determined according to the presenceofHRP reaction product in the graft and the surroun­ding parenchyma.

RESULTS

Cresyl violet (nissl) stained sections demonstratedthe pres ence of both solid and cell suspension graftsof CNS tissue in the ho st corpus callosum at thepredetermined coordinates (Fig. la and c). In cor­responding sections. the presence of AChE-positiveneurons within the feta! grafts was detected by AChEhistochemistry in the ho st rostral corpus caliosum.where ho st cholinergic neurons are usualiy absent(Fig. 1b and d). By this method. both presence of thefetal tissue within the host parenchyma and localisa­tion of the surviving recipient neurons were assess­ed. Detection of HRP reaction product both in thegraft and the surrounding tissue was done underdark field miaoscopy in the corresponding sections.Furthermore. the extent of neovascularization was

observed within the graft tissue and the surroundingbrain outlined by the reaction product in the vessels.

One to three days post-transplantation :

Sections taken on the first postoperative daydemonstrated that the solid tissue grafts were looselyadherent to the adjancent surfaces to some extent.with hemorrhagic debris present. The celi suspensiongrafts on the corresponding sides showed con­siderably less tauma tothe parenchyina comparedwith the solid grafts. By the third day. celis withinthe solid grafts stained weli with aesyl violet. anddemarcation with the surrounding brain was less ob­vious. In corresponding sections which were treatedwith AChE histochemistry. AChE positive neuronswere present only at periphery of the solid grafts in­dicating neuron damage at the central parts. Celi

Turkish Neurosurgery 4: II - 26. 1994 Akalan: Blood-Brain Bariier Changes in Fetal Transplants

Fig. 1 : 50u thiek eoronal seetions through the transplantation site showIng intraparenchymal grafts of fetal CNS tissue in solid from withinthe host corpus ca1losum on the third postoperative day. a) Nissl stained seCtIons where reaetive g1ial aeeumulation is apparent atthe graft-host parenchyma Interfaee (arrow heads). Same infiltration is also present along the eanniua traet (arrow). b) CorrespondingseCtIon treated with AChE mstochemistry. ACh positive neurons witmn the graft appear dark on the right eorner (arrows). Remain­ing parts of the graft contaIn nly "ghosts" of neurons indicating eelluiar breakdown. c) Nissl stained seetion of the contralateralsIde of the host eorpus eallosum that reeeived fetai CNS tissue in eell suspension from. Gliotie reaetion is less apparent comparedto solid grafts. d) AchE mstochemistry for the same seetion demonstrates Ach positive eells distributed eveniy throughout the graftindieating a better survival rate eompared to the solid eounterpart.

suspension grafts. on the other hand. showeduniform distribution of AchE positive neurons in­dieating a rapid reperfusion. Dark field mieroseopyexhibited a massive HRPreaetion throughout the firstto third days. along the needle traet and the grafts.including the adjaeent host tissue (Fig.2a and b). Theleakage was more pronouneed on the solid graft side.indieating the greater surgical trauma exerted by solidgrafting teehniques. Host blood vessels were outlin­ed with HRPreaction produet. No HRPlabelied bloodvessels eould be deteeted in either solid or eeli

suspension grafts. There was no remarkable changein the first and third days of both solid and eellsuspension tissue with respeet to HRP leakage. Onthe third day. less maaophage and glial response waspresent in eresyl violet stained seetions.

Seven to ten days post.transplantation :

By the seventh day the maaophage response wasdeereasing in the surrounding host tissue. HRP reae­tion produet was absent within the parenehyma ofthe solid and eell suspension grafts (Fig. 3a and b).The host-graft interfaee show ed some slight preseneeof reaetion produet in the solid grafts as well as theneedle traets for both sides. Grafts surviving to tendays showed results similar to those of seven days.Viable eells were demonstrated by aesyl violet stain­ing and AchE histoehemistry in corresponding see­tions for both solid and eell suspension grafts. At thistime. reaetion produet was no longer present at thehost-graft interfaee with HRPhistochemistry. Vesselsfilled with reaetion produet eould be traeed topenetrate the eell suspension grafts. At this time.

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Turkish Neurosurgery 4: II - 16. 1994 Akalan: Blood-Brain Barrier Cbanges in Fetal Transplants

Fig. 1: HRP bistochemistry of. a) solid. and b) cell suspension grafts in coronal sedions under dark field microscopy. three days post­grafting. Both grafts and the neigbbouring bost tissue are Eilled with HRP readion product indicating a leaky BBB. Vascu1ature withnormal BBB properties is outlined by HRP readion within the bost parencbyma (arrows).

Fig. 3 : HRP bistochemistry on the seventb day. a) solid. b) cell suspension grafts on contralateral sides of the corpus ca1losum in the samehost. No HRP leakage is detected in the graft tissue (arrow heads) or the neigbouring host parenchyma. Non-leaky vessels witbinthe solid and cell suspension grafts are delineated with the reaction product (arrows). Note the presence of a large caliber vesseloriginating from the host tissue and penetrating the graft (small arrows).

reaetion produet was no longer present at the host­graft interfaee with HRP histochemistry. Vessels mi­ed with reaetion produet could be traeed to penetratethe eelI suspension grafts. Although the solid graftsalso harboured fine ealiber vessels throughout thetissue. alIianee with the host tissue was less eon­

spieuous.

DlSCUSSION

Survival and funetional effeetiveness of in­

traeerebralIy transplanted nervous tissue depend onits rapid reperfusion through the ho st parenehyma(6.8.9.11.17.18.22). Few reports addressing the timeeourse of angiogenesis within intraeerebral solidgrafts are available (1.2.8.9.17.18.28.29).According tothe studies with solid grafts. reperfusion of thegrafted tissue starts within hours and revaseulariza-

24

tion oeeurs within 24-72 hours (8.9.17.18). In ourstudy. similar results were obtained with the solidfetal CNS grafts. whieh were found to be filIed withHRP reaetion produet within 24 hours folIowingtransplantation. when a tissue reeeives its bloodsupply solely by the ingrowth of new vessels as intransplanted neoplasms or in traurnatized brain.neovaseularization does not begin before 3 days(4.6.11.22.28).Therefore. it is speeulated that existingvessels in the solid implants are reperfused ratherthan replaeed by ingrowing host vessels(1.8.17.22.24.28).

Our study was designed to examine the timeeourse of angiogenesis in solid versus eeli suspensiongrafts under similar eonditions. Le.. the same hostreeeived both types of fetal tissue at eorrespondingeoordinates. Our findings suggested that. there was

Turkish Neurosurgery 4: z1 - 26. 1994

no dillerence on the time course of revascularization

of solid and cell suspension grafts. both were foundto be filled with blood-borne HRP within 24 hours.

Since cell suspension grafts did not contain anyvascular elements. the vasculature of the grafts musthave been derived from the host. It is obscure

whether solid grafts are solely reperfused by the in­growing vessels from the host or whether some kindof anastomosis takes place with the ho st and graftvasculature. One finding in this study to support newgrowth in solid grafts is that only neurons at the host­graft interface were found to survive as show n byAChE histochemistry. On the other hand, survivingneurons within cell suspensions were uniformlydistributed which is a staightforward advantage ofthis technique over solid tissue implantation, as dtedelsewhere (8.9.12.13).Nevertheless. the overall heal­ing and neovascularization process was completedby seven to ten days in both solid and cell suspen­sion grafts in our study. At the seventh day follow­ing transplantation. vessels within the grafted tissuewere clearly outlined with the HRP reaction product.

Another crucial aspect for a functioning in­tracerebral neural graft is the status of the BBB. En­dothelial cells lining the capillaries of the CNS havestructural and functional characteristics that con­

stitute a regulatory barrier between the blood andthe brain (25-27.36). This barrier selectively isolatesdrculating compounds in the blood from those pro­duced by the brain providing a homeostatic environ­ment in both the extracellular and cerebrospinal fluidcompartments (31.36). The integrity of the BBB isaltered by neoplasm growth. trauma or stroke.leading to functional and structural disturbances inthe brain tissue (15.34.35). Similar changes apply tointracerebral neural transplantation. The mechanismof BBB disruption following neural grafting can resultfrom both mechanical trauma and angiogenesis pro­voked by the graft (2.5.6.11,18.19.30). Mechanicaltrauma induces a chain of events within the brain,

including the formation of new blood vessels. Newhost capillaries regress to an early developmentalstage of increased permeability (17.35). An alteredBBB accelerates graft rejection by exposing the graft·santigenic structure to the host's blood-borne immunedefensive mechanism. There is an ongoing discussionon the status of BBB following intracerebral transplan­tation of neural tissue (7.8.10.15.28.-30.32). Whilemost studies with solid tissue have demonstrated

reformation of the BBB following transplantation.

Akalan: Blood-Brain Barrier Changes in Fetal Transplants

challenging results are also reported (17.18.28-30).Inour study. solid as well as cell suspension grafts ex­amined at the first and third day s after grafting show­ed a diffuse peroxidase reaction in the graft and thesurrounding tissue. At sevne and ten days. no HRP

reaction product from the grafts was found. indicatingthat the healing and neovascularization processeshad stabilized in both types of graft in the same timesequence. The probable reason for the controversywith studies showing a continuous BBB leakage aftertransplantation is the graft type and the site beingused. Studies suggest that the grafted tissue main­tains its vascular phenotype despite the change in en­vironment (1.8-10. 13.33). Therefore when a CNStissue which originally has impermeable vessels istransplanted in solid from. it is most likely that thoseproperties would withstand in the new location.When non-CNS solid tissue such as adrenal medulla

or superior cervical ganglion is used. originally leakyvessels within the graft are not expected to changetheir vascular phenotype in the host parenchymaresulting in continuous leakage at the graft site. Onthe other land, perfusion after grafting cell suspen­sions totally depends on neovascularization. becausethe enzymatic treatment disrupts the vasculature sothe donor tissue has no inherent vascular structures.

The ingrowing vascular network should maintain itsbarrier properties depending the local environment(3.14.16). This was confirmed in our study: theneovasculature of the cell suspension grafts of CNStissue established a non-Ieaky barrier in the sametime as its solid counterpart. On the contrary. whengrafts are placed in CNS areas close to previntricularorgans or the cortical surface with incomplete BBB.

the graft tissue will have leaky vessels due to the loealenvironment (7). In fact. in studies (17.18.28-30) in­dicating a permanent BBB breakdown followingtransplantation. either non-CNS tissue were used asdonors or the grafts were placed in leaky areas of theCNS.

In conclusion. CNS tissue in either solid or cell

suspension from is rapidly perfused within 24 hoursfollowing intracerebral transplantation. The vascularnetwork with BBB properties is not established forseven days. These results confirm previous studiesthat a solid tissue with BBB properties will reestablishthose properties. indicating blood vessels within thegrafted tissue are preserved and maintain the mor­phological and permeability characteristics theymanifest initially (2.3.6).Moreover. suspension grafts

25

Turkish Neurosurgery 4: 21 ·26. 1994

of the same origin show the same properties undersimilar conditions. According to our data. it can bespeculated that the vascular properties of in·traeerebrally grafted tissue are dietated by the graftitself and the local environment depending on thetransplantation technique used.

Correspondence: Dr. Nejat Akalan.Hacettepe Tip FakültesiBeyin Cerrahisi DepartmaniSihhiye. 06100 Ankara. Türkiyephone: (90) 312 310 84 95Fax: (90) 312 311 II 31

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