Experimental Transplantation of Human Embryonic Cortical Tissue After Vascular Lesion in the Motor Cortex of Bonnet Monkey (Macaca radiata)
Author(s): Kayalvizhi, I.
Vol. 52, No. 1 (2003-01 - 2003-12)
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.
References:
- Borlongan C.V., Koutouzia T.J., Jorden J.R., Martinez, R;
Rodriguez A.I., Poulos S.G., Freeman T.B., Mckeown P.,
Cahill D.W., Nishino H.; Sanberg P.R. (1997) : Neural
transplantation as an experimental treatment modality for
cerebral ischemia. Neuroscience Biobehaviour Review 21
(1) : 79-90.
- Brown A.W. Brierley J.B. (1973) : The earliest alterations in
rat neurons and astrocytes after anoxia ischemia. Acta
Neuropathologica 23 : 9-22.
- Chen H., Chopp M., Schultz L.,Garcia J.H. (1993).
Sequential neuronal and astrocytic changes after transient
middle cerebral artery occlusion in the rat. Journal of
Neurological Science. 118: 109-116.
- Das G.D. (1985) : Development of neocortical transplants.
Quoted in Bjorklund a. and Stenevi U. (Eds.) Neural grafting
in the mammalian CNS. 5 : 101-123.
- Dunnett S.B. (1990) : Review : Neural transplantation in
animal models of dementia. European Journal of
Neuroscience 21: 567-587.
- Emmett C.J., Berg W.J.; Seley P.J. (1990):
Microtransplantation of Neural cells into adult rat brain
Neuroscience. 38 (1) : 213-222.
- Garcia J.H., Lossinsky A.S. Kaufman F.C.; Conger K.A.
(1978): Neuronal ischemic injury : Light microscopic
ultrastructure and biochemistry. Acta Neuropathology (Berlin)
43 : 85-95.
- Grabowski M., Brundin P.; Johansson B.B. (1993):
Functional integration of cortical grafts placed in brain
infarcts of rats. Annals of Neurology 34 : 362-368.
- Hadani M., Freeman T., Munsiff A., Youngs W.; Flamm E.
(1992) : Foetal cortical cells survive in focal cerebral infarct
after permanent occlusion of the middle cerebral artery in
rats. Journal of Neurotrauma 9 : 107-112.
- Hitchcock E.R., Kenny B.G., Clough C.G., Hughes R.C.,
Henderson B.T.H.; Detta A. (1990) : Stereotactic implantation
of fetal mesencephalon : Progress in Brain Research 82 :
723-728.
- Johansson B.B. (1996) : Environmental influence on outcome
after experiment brain infarction. Acta Neurochirugie-
Supplement-Wien 66 : 63-67.
- Macklis J.D. (1993) : Transplanted neocortical neurons
migrate selectively into regions of neuronal degeneration
produced by chromophore-targeted laser photolysis. Journal
of Neuroscience 13 : 3846-3862.
- Mampalan T.J.; Gonzalez M.F., Weinstein P.; Sharp E.R.
(1988). Neuronal changes in fetal cortex transplants to
ischemic adult rat cortex. Journal of Neurosurgory 69 : 904
912.
- Neito-Sampedro M., Whittemore S.R., Needles D.L., Larson
J.; Cotman C.W. (1984) : The survival of brain transplants is
enhanced by extracts from injured brain. Proceedings
National Academy Science. USA 81 : 6250-6254.
- Nishino H. and Borlongan, C.V. (2000): Restoration of
function by neural transplantation in the ischemic, brain Prog
gressive Brain Research 127 : 461-476.
- Onizuka K., Fukuda A., Kunimatsu M., Saski M., Takaku A.;
Nishino H. (1996) : Early cytopathic features in rat ischemia
model and reconstruction by neural graft. Experimental
Neurology. 37(2) : 324-332.
- Poltorak M., Isono M., Freed W.J., Ronnett G.V. (1992):
Human cortical neuronal cell line : Further in vitro
characterization and suitability for brain transplantation. Cell
Transplantation. 1 : 3-15.
- Redmond D.E., Roth R.H., Spencer D.D., Naftolin F., Leranth
C., Robbins R.J., Marek K.L., Elsworth J.D., Taylor J.R.;
Sass K.J. (1993): Neural trasplantation for neurodegenerative
diseases, past present and future. Annals of New York
Academy Science. 695 : 258-266.
- Sorensen J.C., Grabowski M., Zimmer J.; Johansson B.B.
(1996); Fetal neocortical tissue blocks implanted in brain
infarcts of adult rats interconnect with the host brain.
Experimental Neurology. 138(2) : 227-235.
- Tandon P.N., Gomathy G., Mahapatra A.K.; Shetty A.K.
(1990) : Neural transplantation in mammals : our experience.
Proceedings of Indian National Science Academy.
B56 : 51-58.
- Tsymbalik U.I., Kopyov O.V., Picheur L.D., Gordlenko O.V.,
Sherba I.N.; Rudiak K.E. (1991) : Preliminary results of
neurotransplantation in patients with infantile cerebral palsy.
Proceedings Soviet-Indian Symposium on
Neurontransplantation and Developmental Neurology. July 15, Puschino, USSR.
- Williams P.L., Warwick R., Dyson, M.; Bannister L.H. : Gray's
Anatomy. Churchil Livingstone, Medical division of Longman
UK Ltd. New York. (1992)
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.
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.
References:
- Borlongan C.V., Koutouzia T.J., Jorden J.R., Martinez, R; Rodriguez A.I., Poulos S.G., Freeman T.B., Mckeown P., Cahill D.W., Nishino H.; Sanberg P.R. (1997) : Neural transplantation as an experimental treatment modality for cerebral ischemia. Neuroscience Biobehaviour Review 21 (1) : 79-90.
- Brown A.W. Brierley J.B. (1973) : The earliest alterations in rat neurons and astrocytes after anoxia ischemia. Acta Neuropathologica 23 : 9-22.
- Chen H., Chopp M., Schultz L.,Garcia J.H. (1993). Sequential neuronal and astrocytic changes after transient middle cerebral artery occlusion in the rat. Journal of Neurological Science. 118: 109-116.
- Das G.D. (1985) : Development of neocortical transplants. Quoted in Bjorklund a. and Stenevi U. (Eds.) Neural grafting in the mammalian CNS. 5 : 101-123.
- Dunnett S.B. (1990) : Review : Neural transplantation in animal models of dementia. European Journal of Neuroscience 21: 567-587.
- Emmett C.J., Berg W.J.; Seley P.J. (1990): Microtransplantation of Neural cells into adult rat brain Neuroscience. 38 (1) : 213-222.
- Garcia J.H., Lossinsky A.S. Kaufman F.C.; Conger K.A. (1978): Neuronal ischemic injury : Light microscopic ultrastructure and biochemistry. Acta Neuropathology (Berlin) 43 : 85-95.
- Grabowski M., Brundin P.; Johansson B.B. (1993): Functional integration of cortical grafts placed in brain infarcts of rats. Annals of Neurology 34 : 362-368.
- Hadani M., Freeman T., Munsiff A., Youngs W.; Flamm E. (1992) : Foetal cortical cells survive in focal cerebral infarct after permanent occlusion of the middle cerebral artery in rats. Journal of Neurotrauma 9 : 107-112.
- Hitchcock E.R., Kenny B.G., Clough C.G., Hughes R.C., Henderson B.T.H.; Detta A. (1990) : Stereotactic implantation of fetal mesencephalon : Progress in Brain Research 82 : 723-728.
- Johansson B.B. (1996) : Environmental influence on outcome after experiment brain infarction. Acta Neurochirugie- Supplement-Wien 66 : 63-67.
- Macklis J.D. (1993) : Transplanted neocortical neurons migrate selectively into regions of neuronal degeneration produced by chromophore-targeted laser photolysis. Journal of Neuroscience 13 : 3846-3862.
- Mampalan T.J.; Gonzalez M.F., Weinstein P.; Sharp E.R. (1988). Neuronal changes in fetal cortex transplants to ischemic adult rat cortex. Journal of Neurosurgory 69 : 904 912.
- Neito-Sampedro M., Whittemore S.R., Needles D.L., Larson J.; Cotman C.W. (1984) : The survival of brain transplants is enhanced by extracts from injured brain. Proceedings National Academy Science. USA 81 : 6250-6254.
- Nishino H. and Borlongan, C.V. (2000): Restoration of function by neural transplantation in the ischemic, brain Prog gressive Brain Research 127 : 461-476.
- Onizuka K., Fukuda A., Kunimatsu M., Saski M., Takaku A.; Nishino H. (1996) : Early cytopathic features in rat ischemia model and reconstruction by neural graft. Experimental Neurology. 37(2) : 324-332.
- Poltorak M., Isono M., Freed W.J., Ronnett G.V. (1992): Human cortical neuronal cell line : Further in vitro characterization and suitability for brain transplantation. Cell Transplantation. 1 : 3-15.
- Redmond D.E., Roth R.H., Spencer D.D., Naftolin F., Leranth C., Robbins R.J., Marek K.L., Elsworth J.D., Taylor J.R.; Sass K.J. (1993): Neural trasplantation for neurodegenerative diseases, past present and future. Annals of New York Academy Science. 695 : 258-266.
- Sorensen J.C., Grabowski M., Zimmer J.; Johansson B.B. (1996); Fetal neocortical tissue blocks implanted in brain infarcts of adult rats interconnect with the host brain. Experimental Neurology. 138(2) : 227-235.
- Tandon P.N., Gomathy G., Mahapatra A.K.; Shetty A.K. (1990) : Neural transplantation in mammals : our experience. Proceedings of Indian National Science Academy. B56 : 51-58.
- Tsymbalik U.I., Kopyov O.V., Picheur L.D., Gordlenko O.V., Sherba I.N.; Rudiak K.E. (1991) : Preliminary results of neurotransplantation in patients with infantile cerebral palsy. Proceedings Soviet-Indian Symposium on Neurontransplantation and Developmental Neurology. July 15, Puschino, USSR.
- Williams P.L., Warwick R., Dyson, M.; Bannister L.H. : Gray's Anatomy. Churchil Livingstone, Medical division of Longman UK Ltd. New York. (1992)

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.