Peter Høst Poulsen

Xenografting of fetal mouse hippocampal tissue to the brain of adult rats: effects of Cyclosporin A treatment

SummaryThis study examines the effect of the immunosuppressive drug Cyclosporin A (CyA) on the survival and differentiation of solid grafts of fetal (E16–17) mouse hippocampi transplanted to the brain of adult rats. The CyA was given as daily subcutaneous injections of 20 mg/kg from the day before transplantation with reduction of the dose to 15 mg/kg after 14 days. Five weeks after transplantation neuron containing xenografts were recovered in 11 out of 17 CyA-treated recipients (65%). After 8 weeks 9 out of 21 grafts were found (43%). In the control groups, treated only with the vehicle olive oil, 8 out of 14 xenografts were recovered after 5 weeks survival (36%) and only 3 out of 17 after 8 weeks (18%). All xenografts were infiltrated with mononuclear lymphocytic-like cells, but the infiltration was least extensive and least dense in the CyA treated animals. An observed correlation between this cellular infiltration and the gliosis in the xenografts suggested that CyA also directly or indirectly influenced the glial reaction. Most surviving xenografts were located next to the lateral ventricles or the choroid fissure. They were organotypically organized with identifyable cell and neuropil layers, and their connectional organization was similar to rat and mouse allografts grafted to adult recipients. In the absence of major extrinsic afferents the intrinsic pathways observed with Timm staining had reorganized according to known principles for aberrant growth and collateral sprouting. Ingrowth of extrinsic host afferents was only demonstrated for AChE positive host fibers. We conclude that CyA treatment of adult rat recipients can increase the survival of intracerebral fetal mouse hippocampal xenografts and reduce the histological signs of rejection. Xenografting combined with CyA treatment thereby permits the use of a wider spectrum of donor neurons for studies of neuronal interaction and repair.

The DaNeX study of embryonic mesencephalic, dopaminergic tissue grafted to a minipig model of Parkinson's disease: preliminary findings of effect of MPTP poisoning on striatal dopaminergic markers.

A multicenter study is under way to investigate the efficacy of allografting of embryonic mesencephalic neurons in a pig model of Parkinsons disease. We have first established that a stable parkinsonian syndrome can be established by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication of adult male Göttingen minipigs. We are now using positron emission tomography (PET) methods for testing the physiological responses to MPTP intoxication and the time course of the response to several treatment strategies. We now report preliminary results obtained in 11 pigs employed in the initial phase of the study; the completed study shall ultimately include 30 pigs. Animals were randomly assigned to one of five groups: 1) Control, 2) MPTP intoxication, 3) MPTP intoxication followed by allograft, 4) MPTP intoxication followed by allograft with immunosuppression, and 5) MPTP intoxication followed by allograft with immunosuppression and co-grafting of immortalized HiB5 cells, which had been manipulated to secrete glia cell line-derived neurotrophic factor (GDNF) (≈2 ng GDNF/h/105 cells). MPTP was administered (1 mg/kg/day, SC) for 7–10 days until the pigs had developed mild parkinsonian symptoms of muscle rigidity, hypokinesia, and impaired coordination, especially of the hind limbs. Approximately 2 weeks after the last MPTP dose, animals received a T1-weighted magnetic resonance imaging (MRI) scan, and a series of dynamic PET recordings. After the first series of PET scans, four grafts of porcine embryonic mesencephalic tissue (E28 days) were placed in each striatum of some MPTP-intoxicated pigs, using MRI-based stereotactic techniques. Immunosuppression of some animals with cyclosporin and prednisolone began just prior to surgery. Two more series of PET scans were performed at 4-month intervals after surgery. After the last scans, pigs were killed and the brains were perfused for unbiased stereological examination of cytological and histochemical markers in striatum and substantial nigra. The behavioral impairment of the animals (the “Parkinsons score”) had been evaluated throughout the 8-month period. Kinetic analysis of the first set of PET scans has indicated that the rate constant for the decarboxylation of FDOPA in catecholamine fibers was reduced by 33% in striatum of the mildly parkinsonian pigs. The rate of association of [11C]NS-2214 to catecholamine uptake sites was reduced by 62% in the same groups of pigs. No significant difference was found in the binding potential of [11C]raclopride to the dopamine D2-like receptors in striatum of the MPTP-intoxicated versus control pigs. These preliminary results are suggestive that the activity of DOPA decarboxylase may be upregulated in the partially denervated pig striatum.

In vivo estimation of cerebral blood flow, oxygen consumption and glucose metabolism in the pig by [15O]water injection, [15O]oxygen inhalation and dual injections of [18F]fluorodeoxyglucose

There is a need for suitable non-primate laboratory animals for studies of brain function by positron emission tomography (PET). To provide a comparative index of the circulatory physiology of the pig, we have applied novel PET tracer methodology to seven anaesthetized pigs, and measured cerebral regional oxygen consumption (CMR[O2]), cerebral blood flow (CBF), and cerebral glucose metabolism (CMR[glc]). Blood flow and flow-metabolism couple were estimated for selected cerebral regions of interest. We found an average hemispheric CMR(O2) of 171 +/- 18 micromol/100 cm3/min. Individual hemispheric CBF measurements varied between 33 and 41 ml/100 cm3/min, with an average of 37 +/- 3 ml/100 cm3/min at an average PaCO2 of 4.3 +/- 0.9 kPa. The blood flow dependency on arterial PCO2 was calculated from the results of the carbon dioxide response in two pigs in which the CBF measurements obeyed the equation CBF (ml/100 cm3/min) = 8.9 PaCO2 (kPa). In each pig, CMR(glc) was studied twice with a double-injection FDG method. In the first session, the values of CMR(glc) averaged 27 +/- 3 and 23 +/- 4 micromol/100 cm3/min, estimated by multilinear and linear regression analysis, respectively. In the second session, the corresponding averages were 27 +/- 3 and 24 +/- 3 micromol/100 cm3/min, respectively. The average oxygen extraction fraction was 0.46 +/- 0.09 and the oxygen-glucose ratio was 6.1 +/- 0.8. The findings indicate that the pig is suitable for PET studies of cerebral blood flow, cerebral oxygen consumption and glucose metabolism.

On the Oxygenation of Hemoglobin in the Human Brain

We test three hypotheses arising from a model of oxygen delivery to brain tissue. The hypotheses claim that mitochondrial oxygen is negligible in brain tissue such that oxygen consumption depends solely on the mean capillary oxygen tension for a given capillary density; that capillary density is adjusted to satisfy the average steady-state oxygen requirement; and that sudden changes of brain function are subserved by changes of blood flow which adjust the mean capillary oxygen tension in the required direction. The results of the tests did not reject the two former hypotheses and only partly rejected the latter: Sudden changes of blood flow are not always accompanied by increases of oxygen consumption. When they are, changes of apparent capillary density (physiological recruitment) can occur.

Xenografts of mouse hippocampal tissue, exchange of laminar and neuropeptide specific nerve connections with the host rat brain

Immature hippocampal and fascia dentata tissue from embryonic and newborn C57 mice was grafted to the hippocampal region of newborn Kyoto rats. The age of the donor mice varied from embryonic day 13 to the day of birth, and the recipient rats from the day of birth up to 2 days. After survival times of from 5 weeks to 1 year the recipient brains were histologically processed for the tracing of host-xenograft connections by silver staining and electron microscopy of anterograde degeneration, AChE histochemistry, immunohistochemical demonstration of the neuropeptides CCK and enkephalin, and the histochemical Timm sulphide silver method, as well as stained by ordinary cell and fiber stains. The survival of the xenografts depended on the donor age, with less than 10% survival for newborn donors and 60-69% for E13-16 donors. The surviving xenografts developed an organotypic organization and retained a mouse-specific CCK-reactivity in the associational hilodentate system and the dentate mossy fibers. Judged by their positive AChE histochemistry most xenografts received a host rat cholinergic projection when placed in normal cholinoreceptive areas, including areas outside the normal reach of the septo-hippocampal system like the neocortex. Xenografts encroaching on the trajectory of the host rat commissural and perforant path projections or their terminal fields in fascia dentata received laminar and neuropeptide specific host projections. Electron microscopy of host rat perforant path fibers traced to the xenograft dentate molecular layer confirmed the laminar distribution and revealed numerous asymmetric synaptic contacts with spines. An efferent xenograft projection of CCK-reactive mouse mossy fibers into the host CA3 mossy fiber layer demonstrated that this cross-species, mouse to rat innervation also applied to the normal developmental rules, despite the, for the rat abnormal, CCK-content. The formation of laminar and neuropeptide specific mouse-rat nerve connections demonstrates the potentials of intracerebral neuronal grafting in basic and applied neurobiological research by providing new experimental models for the analysis of developmental and functional interactions between nerve cells.

Quantitative PET analysis of regional cerebral blood flow and glucose and oxygen metabolism in response to fenfluramine in living porcine brain

The serotonin agonist fenfluramine has been used widely in humans for studying neuronal activation. We carried out the present study in order to determine whether anesthetized pigs could be used for studying effects of fenfluramine on cerebral functions using positron emission tomography (PET). We obtained quantitative measures of regional cerebral blood flow (rCBF) and of glucose and oxygen utilization (rCMRglc and rCMR(O2)) during intravenous administration of fenfluramine, using [15O]water, [18F]FDG and [15O]oxygen, respectively. Fenfluramine (25 mg/h i.v.) caused a significant rise in rCBF and, to a lesser extent, in rCMR2(O2), but it failed to affect rCMRglc. The findings indicate that quantitative estimation of rCBF by repeated injection of [15O]water was more sensitive than either rCMRO2 or rCMRglc for detecting effects of fenfluramine on serotonin neurotransmission in living porcine brain.

Chapter 34 Xenografts of mouse hippocampal tissue. Formation of nerve connections between the graft fascia dentata and the host rat brain

Publisher Summary This chapter discusses the formation of cross-species nerve connections in mouse hippocampal xenografts placed in the rat brain. The formation of nerve connections between the mouse fascia dentata and the host rat brain appears to be regulated by the same factors, which determines the exchange of host-graft connections in rat allograft studies. The chapter discusses conclusions on the basis of experimental findings: (1) surviving, intracerebral xenografts of mouse fascia dentata and hippocampus are organotypically organized and exchange nerve connections with the host rat brain to the same extent and according to the same principles as rat allografts; (2) complete structural integration in terms of merging of cell and neuropil layers and migration of host rat dentate granule cells into the mouse dentate xenografts is common after grafting to newborn recipients; and (3) the expression of species-specific cholecystokinin (CCK) immunoreactivity in mouse mossy fiber terminals with ingrowth into the host rat hippocampus provides a model for further studies of the developmental and functional interactions between defined groups of neurons.

No influence of the endothelin receptor antagonist bosentan on basal and indomethacin-induced reduction of cerebral blood flow in pigs

Background: The mechanism behind indomethacin‐induced cerebral vasoconstriction is incompletely understood. We tested the hypothesis that the mixed endothelin‐1 receptor antagonist bosentan would modify or prevent indomethacin‐induced reduction of CBF in the anaesthetized pig. Furthermore, we investigated the effect of bosentan on resting CBF and CMRO2.