Journal of the Anatomical Society of India

Department of Anatomy, Dr A.L.M. PGIBMS, Taramani Campus, University of Madras, Chennai. INDIA.

Abstract

A feasibility study of Human Embryonic Cortical Tissue (HECT) as donor in adult bonnet monkey was undertaken. Solid HECT was transplanted in the superficial cortical layers of the motor cortex one hour after devascularisation by stripping the piamateralong with the blood vessels. Transplants survived in all post operative periods (POP) observed. Neuroepithelial and neuroblastic cells inearly periods (18 hrs, 18, 30 and 60 days) were seen in clusters. Most of the grafted cells were viable and exhibited mitotic activity. Aninteresting observation was noticed in graft at 120 days post transplantation. The transplanted tissue was found embedded between laminaII to IV of the host cortex. The grafted mass possessed, differentiated as well as undifferentiated cells. The cellular features in differentiated cells were mature with typical pyramidal neuronal shape similar to the adult host cortical neuron. At the site of needle penetrations as well asin the devascularised host cortex there was glial reaction and mononuclear infiltration at 180 and 240 days after transplantation. There wasdispersion of cells in all directions aided by glial and vascular sproutings, due to this there was decrease in cell density. Neuronal processeswere sprouting from the transplant which integrated with the host cortex. This study shows that HECT in solid fragments exhibits survivalwhen transplanted in devascularised cortical area of bonnet-monkey. This proves that HECT would be suitable for intra-cerebraltransplantation in primates.

Key words: Human embryonic cortical tissue, Devascularised motor cortex, Bonnet monkey.

Introduction:

Neural transplantation provides a technique for the study of brain organisation, development and function in the environment of the host cortex. Successful neural transplantation and interaction of the transplant with the host resulting in functional recovery in various neuro-degenerative disorders has been conducted in experimental animals (Dunnett, 1990; Hitchcock et al, 1990 and Redmond et al, 1993). Even though there are numerous publications in this field to our surprise we found that very few reports were present using rat embryonic intracerebral graft in rat cerebrovascular disorder models (Mampalan et al, 1988; Hadani et al, 1992; Grabowski et al, 1993) as well as in usage of human embryonic neuronal tissue as donor (Poltorak et al, 1992).

Tsymbaliuk et al (1991) succesfully transplanted human embryonic motor cortex into the infarcted area of infantile cerebral palsy patients and showed good improvement in the motor functions in these patients. However, there is no experimental research which could possibly explain the sequence of changes in the graft. So the present work is first of its kind to study the effect of HECT into the devascularised motor area of primate model.

Materials and Methods:

Twelve male Bonnet-monkeys weighing 3.5 to 7.0 Kg were used in this study. Each animal was anaesthetised intraperitonealy using sodium pentobarbitone (30mg/Kg body weight). The head was secured in the stereotaxic frame and about 4 sq. cm piece of frontal bone flap in the left side was reflected by craniotomy. The meninges were incised away from the midline and reflected laterally to expose motor area. With the help of very fine pointed forceps the piamater was stripped along with blood vessels in the motor area (Williams et al, 1992). The animal was maintained for an hour in the same position. After an hour meninges was sutured, bone flap replaced, muscle and scalp sutured in layers. Animals were maintained for various POP. Above procedures were carried out until vascular lesion, using insulin syringe fitted with 26 guage needle loaded with solid fragments (preparation vide infra) of HECT was transplanted intra cortically in 4 or 5 sites at a depth of 2 to 3mm after an hour of vascular lesion which served as vascular lession and transplanted model.

Out of the twelve animals, one animal served as control, two animals were used for studying changes due to vascular lesion i.e. one hour and 240 days after vascular lesion. Nine animals were utilized for producing vascular lesion followed by transplantation. The animals were sacrificed one at a time at intervals of 18 hrs, 18, 30, 60, 120, 180, and 240 days after transplantation. Two animals in 60 and 120 days period had unexpected death. So the experiment was repeated for the above periods.

Preparation of Donor tissue:

The human embryos ranging from 10 to 16 weeks of gestational age were collected (The protocol of the work has been approved by the ethical committee and necessary permission had been obtained from the Director of Medical Education, Tamil Nadu. The consent of the donor mothers to utilize their fetal material for the research purpose was also obtained) in sterile lactated saline after Medical Termination of Pregnancy (MTP) in the maternity hospital and transported within an hour for isolation and fragmentation of presumptive motor area in sterile condition clearing meninges and blood vessels, Fragments were left for culture around 4 to 18 hrs. in minimal essential medium (MEM) and were used for transplantation.

After various POP, the animals were sacrificed by transcardial perfusion under barbitone anaesthesia. After perfusion the brain was dissected and preserved in 10% formalin. The motor area of the left cerebral cortex was processed for routine paraffin sectioning. Sections were cut at 10m thickness of 1/8th serial and stained with Cresyl Fast Violet (CFV), Glees modification of Bielschowsky's method (Silver) and Mallory's Phosphotungstic acid heamatoxylin (PTAH).

Results:

In all animals there was only a transient deficit in the usage of fingers of the right hand to pick small objects. The deficit disappeared over a period of 10 days in all animals. After an hour of vascular lesion, the sections of motor cortical area showed neuronal loss and disorientation of neurons. The outermost layer of grey matter appeared thicker due to oedema whereas neurons in deeper cortical layers were shrunken, angulated, darkly stained and nucleoli were indistinguishable. Neurons also showed microvascularisation in the cytoplasm as well as in the nucleus. In PTAH stained slides numerous swollen astrocytes were encountered in the host cortex. The histopathological changes observed after 240 days of vascular lesion were similar to the lession effects of early hours. The astrocyte proliferation and paired astrocytes were observed in PTAH stained sections.

Histological changes after vascular lesion and transplantation:

In the early stages i.e. 18 hours after transplant, the neural grafts showed a healthy appearance and they were found in an undifferentiated state embedded in host brain. The neuroepithelial cells were held together in their embryonic tissue matrix. A few neuroepithelial cells started proliferating and formed rosettes.

At 18 days post transplantation the HECT were present in the deeper lamina of grey matter and also in white matter (Fig.1) The grafted neurons appeared oval or spindle shaped and did not possess the typical shape of a neuron showing that these cells were still immature. Some cells exhibited mitotic figures indicating proliferation. There were some cell debris and cavity formation inside the transplant where leukocytes were also present. The processes from the immature neurons were evident in silver preparation (Fig. 2).

At 30 days post transplantation the graft was stuck to the surface of the host cortex. The graft neurons were found in irregular clumps, still exhibiting rosettes and very few mature polygonal cells were noted. Grafted neurons possessed processes inter connected with each other as well as with the host cortex.

At 120 days post transplantation, the transplant extended from lamina II to IV, (Fig.3). The HECT in the periphery had differentiated and migrated showing various stages of maturation (Fig.4). These maturing cells formed neuropil with the host cortex. Very few cells were found in the matured state showing the typical shape of the pyramidal neurons and majority of the cells were binucleated (Fig.5 & 6). The grafted neurons which were differentiated showed an attempt to form laminar pattern.

In the late phase i.e. 180 and 240 days after transplantation, the cells were scattered irregularly and their migration resulted in vacuole formation. There was extensive glial reaction and very few cells were retained to the site of transplantation. The neural elements were fusiform in shape and a few pyramidal and oval cells were also present.

In the transplanted animals the host cortex during early phase showed severe necrotic changes in laminae I to III, whereas deeper laminae IV to VI were less affected. In the late phase the ischaemic cells disappeared resulting in a shallow depression in the upper laminae. Numerous astrocytes were encountered, cytoplasm of neurons showed chromatolytic changes and microvacoulation. The nucleus was swollen and showed 4 to 5 vacuoles giving a cart-wheel appearance.

Discussion:

The cortial lesion by pial stripping is one of the methods of devascularisation (Williams et al, 1992). The advantage of using primate rather than rat is to have closer similarity to man in their cerebral vasculature.

Even though cerebral motor cortical area was devascularized, only transient deficits were noted which disappeared within a few days of POP. However, notable histological changes were found which were very similar to those obtained by other workers after anoxic and ischaemic treatment (Brown and Brierley, 1973; Garcia et al, 1978; Chen et al, 1993).

The neuroepithelial cells aggregate to form rosettes within 18 hours of POP and start to divide. Reduction in the number, of rosettes in 30 days shows that neuroepithelial cells disperse to form neuroblasts. Presence of rosettes in 120 days of POP indicates that mitotic activity probably continues till 120 days. Similar observations were also reported by Das (1985) in embryonic rat cortical tissue transplanted in 15 to 20 days old host rat.

Successful transplantation in bonnet-monkeys shown in the present report substantiates the presumption of Tandon (1990) who mentioned that in lesser evolved sub-human primates such as bonnet- monkey, the failure rate is not as high as in Macaca rhesus.

Non selective lesioning methods such as ischemic lesion and surgical ablation do not appear to allow migration, integration of the transplant in the cereberal cortex and cerebellum (Macklis, 1993). However, we have found better integration of the transplant in the cerebral cortex of bonnet-monkey under ischemic conditions in our study. Better survival and integration of the transplant in the host cortex reported in our study correlates to the reports of Mampalan et al, 1988; Hadani et al, 1992; Grabowski et al, 1993; Onizuka et al, 1996; Sorensen et al, 1996; Johansson, 1996; Borlongan et al, 1997; Nishino & Borlongan, 2000 where the embryonic cortical tissue and animal model were rats.

Transplantation is reported to be successful only when the transplant is grafted between 7 days to 14 days after the lesion. Transplanting before or after this interval does not provide successful transplants (Neitro-Sampedro et al, 1984). However, in our study the transplantation performed as early as one hour after vascular lesion has also provided appreciable survival and integration of the transplant.

According to Tandon (1990) successful transplant in monkey was obtained with fetal tissue cryo preserved for four days in culture media in comparison to the high failure rate of fresh foetal tissue transplant. In our study the short term culture of the foetal neural tissue (4 to 20 hours) produced successful transplant.

With conventional transplantation methods used in rats; reaction of necrosis, degeneration, differentiation and hemorrhage are inescapable consequences of implantation (Emmett 1990). In our study the method of implantation used was by routine hypodermic syringe. Due to careful handling and precautions to minimise trauma to host cortex, the transplant proved to be successful causing minimal reaction of necrosis. It may be probably also due to the larger size of the monkey brain in comparison to that of the rat brain.

To conclude as the pial stripping is confined to a small motor area, the functional deficit is transient. However, the histological changes in the pial stripped cortex correlate with the ischemic cell changes produced by arterial occlusion.

HECT is able to withstand the process of preparation and subsequent implantation into the brain of a heterospecific host cortex in bonnet-monkey. HECT grows and integrates with the surrounding devascularised host cortex. In the early phase of transplant the cells were viable, immature and exhibited rosettes. The presence of mitotic division and binucleated neurons were observed at 120 days after transplantation. In the long term survival of transplant, there was decrease in the number of neuronal cell groups at the site of transplantation in comparison to the early phase which might be due to migration of the graft. Solid fragments of HECT used in the present study gave better results. There was sharp increase in degenerating cortical neurons after devascularisation which was reduced in experiment in which devascularisation was followed by transplantation of HECT.

Our preliminary results suggest that HECT could serve as donors for ischemic cortex of primates which may in future be implicable as a new therapeutic measure in Human Stroke Patients. In the present study no immunosuppressive drug was utilized and successful transplant has been observed consistent with the result of Poltorak et al (1992).

Acknowledgements:

We thank Lady Tata Memorial Trust for the financial support to undertake this research work. We acknowledge The Director of Medical Education, Tamilnadu; Director of Institute of Obstetrics and Gynecology, Egmore Chennai; for permitting the collection of the fetal materials.

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Fig. 1

Photomicrograph showing HECT after 18 days of devascularisation and transplantation. The grafts cells stuck to the inner surface of the cavity in the recipient host cortex. CFV stain (200 m).WM - White matter, GM - Grey matter, TR - Transplant, C - Cavity.

Fig. 2

Photomicrograph showing the growth of nerve fibre from the transplant to the host cortex after 18 days of devascularisation and transplantation. Silver stain. (100 m). (TR-Transplant; P-Process)

Fig. 3

Photomicrograph showing HECT present in 120 days of devascularised and transplanted motor cortex. The graft extend from lamina II to IV. CFV stain (200 m).
(TR-Transplant; HC- Host Cortex)

Fig. 4

Photomicrograph showing HECT present in 120 days of devascularisation and transplantation in the host cortex. The grafts were of two distinct groups, differentiated and undifferentiated groups. CFV stain (100 m). DNB - Differentiated neuroblast, UDNB - Undifferentiated neuroblast.

Fig. 5

Photomicrograph showing differentiated neuroblast after 120 days of devascularisation and transplantation. CFV Stain (100 m). NB - Neuroblast.

Fig. 6

Photomicrograph showing HECT after 120 days of devascularisation and transplantation. The neuroblasts were under varying stages of differentiation. Majority were round, fusiform and polygonal in shape. Typical pyramidal neuron was also seen. CFV stain (50 m).
PYN - Pyramidal neuron. BN - Binucleated neuron, PG - Polygonal.