Visualisation of the medial longitudinal fasciculus using fibre tractography in multiple sclerosis patients with internuclear ophthalmoplegia.

BACKGROUND: This study investigates the use of fibre tractography to facilitate visualisation of the medial longitudinal fasciculus (MLF) and the impact of internuclear ophthalmoplegia (INO) causing lesions on these reconstructions of the tract. Improved visualisation of such tracts may improve knowledge, understanding and confidence related to neurological conditions. AIMS: To explore the use of fibre tractography for the visualisation of the MLF in patients with INO. METHODS: Twelve MS subjects with clinical evidence of INO and 12 matched controls underwent magnetic resonance imaging (MRI), including diffusion tensor imaging (DTI), of the brain. Fibre tractography reconstructions were then evaluated and validated by an experienced neuroanatomist. RESULTS: The evaluating neuroanatomist confirmed that the MLF had been reproduced in all of the reconstructed cases (fibre tractography was unsuccessful in five cases). The sensitivity of fibre tractography to MLF pathology was 58.3 % while the specificity was much higher at 85.7 % with a positive predictive value of 87.5 % and a negative predictive value of 54.6 %, with excellent intra-reader reliability. CONCLUSION: This study demonstrates that fibre tractography of the MLF can potentially be performed with a view to facilitating improved visualisation of the tract and associated pathology in cases of INO. This may help explain the association between lesion type and location with clinical symptomatology and may assist in monitoring disease progression. These reconstructions may provide a valuable addition to the teaching and understanding of clinical signs related to subtle pathology.

Autoradiographic analysis of effects of 5-bromodeoxyuridine on neurogenesis in the chick embryo spinal cord.

Bromodeoxyuridine (BUdR) can reversibly inhibit the terminal differentiation of embryonic cell types when they are grown in culture. The goal of these experiments was to see if BUdR could interfere with the terminal differentiation of neurones in the intact chick embryo, a possibility disputed in different reports. Therefore, 0.02 mg BUdR plus 15 microCi [3H]BUdR was injected into the albumen of incubating eggs at stage 14-16 of development. Controls received an equimolar amount of [3H]thymidine ([3H]TdR). The doses and mode of administration are known to result in availability times for the nucleosides that are longer than one cell generation cycle and therefore similar to the pharmacokinetic conditions possible in vitro. The time of treatment is known to correspond to the period of most rapid neurone production in the chick spinal cord. By autoradiography of semi-thin sections the fates of the cells that had incorporated the nucleosides could be followed. BUdR was taken up by the S phase population of the spinal cord neuroepithelium (NE). In the first 10 h after treatment the BUdR treated NE behaved the same as the control. From then until 24 h after treatment, NE cells underwent necrosis as a result of BUdR and neurone production was almost completely suppressed. Between 24 and 48 h after treatment the BUdR-treated NE produced neurones at a faster rate than the TdR-treated controls. However, this effort at compensation was not entirely effective and by 6 days after treatment the BUdR-treated embryos had an absolute reduction in motor neurone number. Motor neurones with BUdR incorporated in their DNA that survived until after neurogenesis was completed were strikingly more lightly labelled than those in controls treated with [3H]TdR. This suggests that the survivors had incorporated less BUdR than those that had died. It was concluded that the neuronal deficit resulting from BUdR treatment was not the result of an inhibition of terminal differentiation, but rather of cytotoxicity.

Anatomy of the extraneural blood supply to the intracranial oculomotor nerve.

AIMS: An anatomical study was undertaken to determine the extraneural blood supply to the intracranial oculomotor nerve. METHODS: Human tissue blocks containing brainstem, cranial nerves II-VI, body of sphenoid, and associated cavernous sinuses were obtained, injected with contrast material, and dissected using a stereoscopic microscope. RESULTS: Eleven oculomotor nerves were dissected, the intracranial part being divided into proximal, middle, and distal (intracavernous) parts. The proximal part of the intracranial oculomotor nerve received extraneural nutrient arterioles from thalamoperforating arteries in all specimens and in six nerves this blood supply was supplemented by branches from other brainstem vessels. Four nerves were seen to be penetrated by branches of brainstem vessels and these penetrating arteries also supplied nutrient arterioles. The middle part of the intracranial oculomotor nerve did not receive nutrient arterioles from adjacent arteries. The distal part of the intracranial oculomotor nerve received nutrient arterioles from the inferior cavernous sinus artery in all 11 nerves and in seven nerves this was supplemented by a tentorial artery arising from the meningohypophyseal trunk. The inferior hypophyseal artery arose from the meningohypophyseal trunk in all 11 cavernous sinuses dissected. CONCLUSION: This study shows a constant pattern to the blood supply of the intracranial oculomotor nerve. It also highlights the close relation between the blood supplies to the intracavernous oculomotor nerve and the pituitary gland.

The effect of low level lead exposure on the postnatal structuring of the rat cerebellum.

Exposure of rat pups to lead (less than 45 micrograms/dl blood) caused no alterations in the rate of cerebellar cell acquisition, migration or final number when compared with age-matched controls. The rate of DNA biosynthesis was higher in the lead-exposed animals compared to controls and remained elevated until postnatal day 10. This observation suggests lengthening of the cell cycle. The expected increase in DNA biosynthetic rate was delayed in neuron-enriched fractions obtained from lead-exposed pups. This may have been due to the isolation procedure specifically selecting internal granular layer cells which migrated precociously during a protracted G1 phase in a lead-impaired cell cycle. Morphometric studies revealed no difference in the number, viability and migration of cells within and from the external granular layer of the cerebellum of lead-exposed animals. It is concluded that chronic low level lead exposure has no significant effect on the early structuring of the developing cerebellum.

The structure and function of the ureter in the dog.

THE structure of the ureter was investigated by the examination of sections taken from the ureters of 14 dogs. Simultaneous recordings of peristalsis under conditions of varying urine flow were made from the middle and lower segments of the ureters of 10 dogs under anaesthesia by means of a method that allowed free urine flow.The ureter was found to possess three layers of muscle in its wall, an inner and outer longitudinal layer and a middle layer of fibres whose orientation varied from circular to oblique. On the basis of the arrangement of these three layers the ureter can be divided in its length into three segments. The middle segment has the most completely circular fibres in its middle layer and the junction between it and the upper and lower segments are marked by a change in the orientation of the middle layer fibres from circular in the middle segment to oblique in the upper and lower.Evidence is provided by pressure measurements that these changes provided the basis for sphincteric mechanisms at high rates of urine flow.

Evaluation of a miniature antimony electrode for measurement of myocardial pH.

Myocardial pH reflects the metabolic status of the heart and pH monitoring is an invaluable way to monitor the efficacy of myocardial protection during cardiac surgery. We developed a miniature antimony electrode for pH measurement in the heart. We examined the sensitivity, accuracy and the effects of temperature and oxygen tension on pH readings with this electrode in standard buffers and in anaesthetized dogs. In buffers the antimony electrode exhibited a gradient of -50.3 +/- 1.8 mV pH-1 at 25 degrees C, close to the Nernstian slope and showed a high correlation with conventional glass electrode readings (mean difference 0.027 +/- 0.0035 pH, r2 = 0.97). With increasing temperature the antimony electrode pH readings increased by 0.03 +/- 0.002 pH degree C(-1). With increasing PO2 the pH reading decreased (-0.73 pH/log PO2 mm Hg, r2 = 0.96). In the dog heart the antimony electrode showed a decrease in myocardial pH with increasing PCO2, and an increase in pH when NaHCO3 was given intravenously. Coronary occlusion resulted in paradoxically higher pH readings with the antimony electrode due to the effect of lowered myocardial PO2 interfering with pH measurement. The dissolution of antimony from the electrode in blood plasma was tested and found to be low. These studies suggest that antimony electrodes have low toxicity and provide accurate pH determinations under conditions of constant PO2. For more widespread clinical application, the problem of oxygen interference needs to be solved.

Stereology of the placenta in type 1 and type 2 diabetes.

OBJECTIVE: To assess by stereology the placental structure in type 1 (T1DM) and type 2 (T2DM) diabetic pregnancies compared to normal non-diabetic (ND) controls. STUDY DESIGN: Prospective case control study. Placentae were sampled in a systematic random fashion. Stereological analysis was performed using a computerised stereology programme (Image Pro 6.2, Media Cybernetics, Inc, Silver Spring MD, USA). Participants were matched for gender of infant and mode of delivery. MAIN OUTCOME MEASURES: Volume, length and surface area of placental components; clinical outcome. RESULTS: Ten ND, eight T2DM and ten T1DM women consented to the study. There was no difference between the groups regarding maternal age, neonatal birth weight, or placental weight. On stereological examination, terminal villous volume was significantly increased in both diabetic groups compared to ND controls. Capillary volume and length was increased in T1DM pregnancies compared to ND and T2DM. Capillary length was increased in both diabetic groups compared to ND. When all diabetic groups were compared based on severity of glycaemia those with poor glycaemic control (HbA1c>7%) had higher placental capillary volume than those with good glycaemic control. CONCLUSIONS: This study demonstrates an association between maternal diabetes and increased terminal villous volume. Additionally capillary volume and length is increased in the placentae of normally grown infants of T1DM diabetic mothers compared to non-diabetic controls. Maternal glycaemia appears to influence capillary, but not stromal, development. This suggests that factors other than glycaemia have a role in placental development in pre-gestational diabetes.

The Adriamycin rat/mouse model and its importance to the paediatric surgeon.

The teratogenic effect of Adriamycin (doxorubicin) in the rat model, and more recently in the mouse, has provided paediatric surgeons with a reliable, easily reproducible method of studying the embryology and molecular biology for a range of complex congenital anomalies. Concomitantly these animal models have stimulated interest among embryologists for the effect on the notochord, shedding more light on the important organizational role of this structure in the developing embryo. Finally, as more is learnt of the pathogenesis of the various malformations induced by Adriamycin, future therapeutic interventions involving gene therapy, drugs or surgery may arise. This article reviews the establishment of the Adriamycin rat and mouse models, examines their impact on various congenital malformations, and suggests targets for further research.

Differences in nitrergic innervation of the developing chick cloaca and colorectum.

The intrinsic innervation of the developing gut has long been a subject of investigation, but little is known regarding that of the embryonic cloaca. The cloaca, like the rest of the gastrointestinal tract, is intrinsically innervated by the enteric nervous system. Nitrergic neurons and fibres make up a large part of this system, thus, their distribution provides us with a useful insight into its development. Cloacal and colorectal tissue specimens were removed from chick embryos at embryonic days 11 (E11), E15 and E19. NADPH-diaphorase (NADPH-d) histochemistry was carried out using whole mount tissue preparations. Ganglia density, the number of NADPH-d-positive cells per ganglia in the myenteric plexus and cell size were calculated and statistical analysis was performed to compare both regions of the gut (P<0.001). There were significant differences in the ganglia density in the cloaca compared to the colorectum at E11 (P<0.05) and E15 (P<0.01), with the colorectum having a much denser network. In both the cloaca and the colorectum, ganglia density significantly decreased with age (P<0.001), while significant differences were observed in the number of NADPH-d-positive cells per ganglia in both regions through development. Total cell size was similar in both the cloaca and colorectum at each stage and increased in both regions through development, predominantly due to an increase in the cytoplasm. Results reveal striking differences in innervation between the chick embryo cloaca and colorectum. The sparse network of innervation evident within the cloaca in contrast to the dense network within the colorectum emphasizes the individuality of both regions. These results highlight the need for a further in-depth analysis of the enteric nervous system's development within the embryonic cloaca.

The effect of vagal neural crest ablation on the chick embryo cloaca.

The cloaca, the caudal limit of the avian gastrointestinal tract, acts as a collecting chamber into which the gastrointestinal, urinary, and genital tracts discharge. It is intrinsically innervated by the enteric nervous system, which is derived from neural crest emigres that migrate from the vagal and sacral regions of the neural tube. Abnormal cloacal development can cause a number of anorectal anomalies, including persistent cloaca. Ablation of the vagal neural crest has previously been shown to result in an aganglionic hindgut to the extent of the colorectum. The aim of our study was to investigate the effect of vagal neural crest ablation on the cloaca, the limit of the hindgut in the developing chick embryo. Chick embryos were incubated until the 10-12 somite stage. The vagal neural tube corresponding to the level of somites 3-6 was then ablated, and eggs were incubated until harvested on embryonic day 11 (E11). Whole chick embryos were fixed, embedded in paraffin, and sectioned. Immunohistochemistry was then carried out using the HNK-1 monoclonal antibody to label neural crest cells, and results were assessed by light microscopy. Vagal neural crest ablation resulted in a dramatic decrease in the number of neural crest cells colonizing the chick embryo cloaca compared with control embryos. Ablated embryos contained only a small number of HNK-1-positive neural crest cells, which were scattered within the myenteric plexus in a disorganised pattern. Hypoganglionosis was also evident in other regions of the hindgut in ablated embryos. Ablation of the vagal neural crest results in a hypoganglionic cloaca in addition to hypoganglionosis of the hindgut. These results suggest that the cloaca is largely innervated by vagal neural crest emigres. Further studies involving quail-chick chimeras to investigate the exact contribution provided by both vagal and sacral neural crest cells to the cloaca should increase our understanding of the pathophysiology of conditions like persistent cloaca.

The effects of 5-bromodeoxyuridine on fusion of the cranial neural folds in the mouse embryo.

The effects of 500 and 300 mg/kg bromodeoxyuridine (BUdR) on the process of fusion of the neural folds were tested after injection into pregnant mice on day 8 of gestation (192 hours postcoitum). Various doses of the natural nucleoside, thymidine (TdR), were also tested. Both doses of BUdR retarded growth to the same extent, but only the larger dose caused neural tube defects in 28.8% of embryos. Treatment with the larger dose also caused extensive cell necrosis to appear in the neuroepithelium of the neural folds between 12 and 15 hours after treatment. No changes were detectable with the light microscope up to this time. Measurement of the cell generation time in treated and control embryos indicated that the BUdR prolonged the cycle by about 2 hours and that the dying cells were in the second DNA synthetic phase following incorporation of the analog. Treatment with the smaller dose of BUdR caused minimal cell necrosis. This was taken as evidence for the importance of cell necrosis in the pathogenesis of BUdR-induced neural tube defects. Treatment with excess TdR did not cause either neural tube defects or cell necrosis, and a dose of TdR equimolar with the large dose of BUdR (400 mg/kg TdR) did not retard growth. Doses of 800 and 1,200 mg/kg TdR retarded growth to the same extent as BUdR. The administration of an equimolar amount of TdR, along with the teratogenic dose of BUdR, prevented the occurrence of cell necrosis and neural tube defects. When treatments were given on day 9 of gestation, 500 mg/kg BUdR caused cell necrosis in the neuroepithelium about 15 hours after treatment but no neural tube defects were produced by day 9 after treatment. It is suggested that in this case cell necrosis occurred too late to interfere with neural fold fusion. It was concluded that the ability of BUdR to cause exencephaly in mouse embryos was due to cell necrosis in the neuroepithelium.

Ethanol teratogenicity in mice: an electron microscopic study.

In this study, the neuroepithelium (NE) cells of the mouse embryo were examined with the electron microscope at various intervals after maternal injection of 0.03 ml/g body weight 25% (v/v) ethanol on day 9 of gestation (plug day = day 1), by the intraperitoneal route. Within 1 hour of treatment, the mitochondria of the NE cells became greatly swollen but could recover. Recovery occurred in two phases: a rapid one during the second hour after treatment, followed by a more gradual one that lasted until 12 hours after treatment. About 5 hours after treatment, dying and fragmenting cells were seen in the NE of all embryos examined. The debris from this necrosis was phagocytosed by neighbouring healthy cells. Also at 5 hours after treatment there was an apparent expansion of the intercellular space of the NE and an enlargement of the apical pseudopodial processes of the NE cells. The latter two changes may have been the result of failure of energy-dependent cell fluid homeostasis consequent to mitochondrial dysfunction. All of these changes were reversed by 15 hours after treatment. Although all embryos examined had abnormalities of the NE, including cell necrosis, at 24 hours after treatment only 28% had failed to complete neural tube formation. Hence, either the degree of ethanol-induced damage varies between embryos in the same litter, or the sensitive period is so restricted that variations in stage of development within a litter can account for the lack of concordance between the presence of cellular damage and the subsequent occurrence of a neural tube defect.

The cellular effect of 5-bromodeoxyuridine on the mammalian embryo.

It is well known that 5-bromodeoxyuridine (BUdR) when injected into pregnant animals may cause exencephaly, cleft palate, and limb abnormalities. Similarly, it is well established that the drug when added to a culture medium may prevent differentiation of embryonic cell systems without affecting cell division or cell viability. The goal of our experiments was to examine whether the congenital malformations resulting from BUdR treatment were due to lack of differentiation of certain cell lines or were due to other mechanisms. The effects of BUdR on proliferating and differentiating cells in the 12-day mouse embryo were therefore examined and special attention was given to the proliferating cells of the rhombic lip which give rise to the Purkinje cells. When the embryos were treated with BUdR the mitotic index of the neuroepithelium of the rhombic lip doubled in value 3 h after treatment and remained high until 24 h later. By using the colchicine index it was calculated that the mitotic duration in the BUdR-treated embryos lasted at least 2 h and that in the control embryos less than 1 h. When the cell generation time in the BUdR treated animals was calculated the length of the S-phase was increased by about 50%. It was thus concluded that BUdR caused an increase in the duration of the S-phase and mitosis, together making the cell cycle 5 h longer than normal. Eighteen hours after treatment many neuroepithelial cells became degenerative. By radioautography it was demonstrated that the degenerating cells were in their second DNA-synthetic phase following BUdR injection and that cells which incorporated BUdR and were differentiating into neurons were not affected. By injecting [3H]BUdR it was found that many cells which incorporated the analogue were able to leave the proliferative population after their first cell division. They migrated to the periphery where they developed into apparently normal Purkinje cells. The additive effects of cell death and retardation of the cell cycle caused a 15% deficit of Purkinje cells in the postnatal cerebellum but the BUdR did not interfere with their differentiation. Thus, contrary to the BUdR effect on cultures of embryonic cells, in vivo the drug causes cell death and a delay in the cell cycle time. Our experiments therefore seem to indicate that the congenital malformations caused by BUdR in the mammalian embryo are caused by cell death and growth retardation rather than by interference with the process of differentiation.

Effects of cadmium on formation of the ventral body wall in chick embryos and their prevention by zinc pretreatment.

BACKGROUND: Cadmium (Cd) is an established experimental teratogen whose effects can be reversed by pretreatment with zinc. Mesodermal development is a frequently reported target for Cd teratogenicity. The aim of this study was to examine the mechanisms of Cd induced body wall defects in chick embryos. METHODS: Chick embryos in shell-less culture were treated with 50 microl of cadmium acetate (8.9 x 10(-5) M Cd(2+)) at 60-hr incubation (H.-H. stages 16-17). Controls received equimolar sodium acetate. Other embryos were treated with various concentrations of zinc acetate and then with Cd or NaAc 1 hrs later. Development was evaluated 48 hrs later. Resin-embedded 1-microm sections were examined at earlier stages. RESULTS: Cd caused embryolethality (35%), ventral body wall defect with malpositioned lower limbs (40%), and weight reduction in survivors. After 4-hr treatment with Cd, breakdown of junctions between peridermal cells with rounding up and desquamation occurred. Shape changes were also seen in the basal layer of the ectoderm. At 4 hr, cell death was evident in lateral plate mesoderm, somites, and neuroepithelium; the lateral plate mesoderm began to grow dorsally, carrying the attached limb buds with it. Zn pretreatment protected against the lethal, teratogenic, and growth-retarding effects of Cd, as well as ectodermal changes and cell death. CONCLUSIONS: Cd disrupts peridermal cell adhesion and induces cell death in the mesoderm. This may result in abnormal growth of lateral plate mesoderm and in a body wall defect. Zn pretreatment prevents both the gross teratogenic effects and the cellular changes, most likely by competition with Cd.

Ethanol teratogenicity in mice: a light microscopic study.

The objective of this study was to see what, if any, cellular changes occurred in the mouse embryo following a single injection of ethanol, a known teratogen in humans and animals, on day 9 of gestation. No changes were seen until 6 hours after injection, when many degenerating cells and necrotic fragments were seen in the neuroepithelium of the neural groove and of the neural tube. In addition, large clear vacuoles were seen in the cytoplasm of many cells and the pseudopodia at the luminal side of the neural groove appeared swollen. The cytoplasm of the latter also contained vacuoles. When tritiated thymidine was injected 5 hours after ethanol and 1 hour before sacrifice, many degenerating cells were labelled. In addition, many cells with labelled nuclei had abnormal vacuoles in the cytoplasm. Hence, it is likely that the toxicity of ethanol is exerted primarily on some component of the cytoplasm and not on DNA synthesis. Twelve hours after ethanol, the cytoplasmic vacuoles and swollen pseudopodia had disappeared, but dying cells were still evident. By 24 hours, the necrotic debris had been completely phagocytosed by healthy neuroepithelial cells. By 50 hours, the neuroepithelium had been cleared of cell debris, although many ethanol-treated embryos had open defects of the cranial neural tube. Treatment of pregnant mice with single doses of acetaldehyde, also an established teratogen in animals, did not produce any cellular changes. However, a single dose of acetaldehyde is rapidly metabolized by the mother, and would not be comparable to the small but continuous blood levels that a dose of ethanol would produce. Hence, we could not conclude with certainty that the cytotoxic effects of ethanol were exerted directly.

The effects of lithium on neurulation stage mouse embryos.

The aim of this study was to evaluate the maternal toxicity and teratogenicity of lithium following intraperitoneal injection (i.p.) with lithium carbonate (Li2CO3) in pregnant CD-1 mice at the developmental stage of neurulation (E8; day of vaginal plug, E0). Light (LM) and electron (TEM) microscopic studies were also done to document the tissue and cellular changes occurring in embryonic tissues during the 48 h following treatment with 300 mg/kg body wt. Li2CO3. Controls were untreated or given equimolar amounts of NaCl or Na2CO3. A pharmacokinetic study showed that lithium was rapidly absorbed from the peritoneal cavity after the above-stated dose, achieved peak serum levels of 9.8 mmol/l within 1 h, had a half-life in the blood of 5 h and was completely cleared by 16 to 24 h after injection. Doses of Li2CO3 > 300 mg/kg body wt. were toxic to adult CD-1 mice. The latter dose had no detectable maternal toxicity but caused a 19% resorption rate and 2% incidence of open cranial neural tube defect in gestations terminated on E18. The malformation and resorption rates in gestations terminated on E11, E12 and E14 were not significantly different from those of E18. A strong litter effect was seen both for the resorption and malformation rates at all stages examined. At 3 h after treatment cell death became evident in the neuroepithelium. Cells continued to die for approximately 17 h and all necrotic debris had been cleared by 48 h. Also at 3 h after treatment small densely stained inclusions began to appear in mesodermal cells. TEM showed these to be non-membrane bound with an irregular shape and variable size; the lack of staining for acid phosphatase indicated a non-lysosomal structure; the ultrastructural features suggested a lipoid basis. At 24 h after treatment vascular ruptures and surface ectodermal ruptures were seen in the cranial mesoderm. These ruptures with extravascated blood were also seen at 48 h after treatment. A litter effect was also noted with respect to the tissue and cellular changes. These experiments suggest that the developing vascular system may be a target for lithium. In addition, the possibility is discussed that lithium induced cell death in the neuroepithelium may lead to neural tube defects.