In situ biodistribution and residency of a topical anti-inflammatory using fluorescence lifetime imaging microscopy.

BACKGROUND: GSK2894512 is a topically delivered investigational drug being developed for treatment of atopic dermatitis and psoriasis. OBJECTIVES: To investigate, in a phase I clinical trial, the spatial biodistribution and residency of GSK2894512 within the epidermis and dermis of healthy human participants noninvasively using fluorescence lifetime imaging microscopy (FLIM). METHODS: Two topical drug formulations containing GSK2894512 1% were applied to the right and left forearms of six participants for seven consecutive days, followed by seven days of observation for residency. FLIM images were obtained daily throughout the study, approximately every 24 h. During the treatment phase of the study, images were collected from each participant pretreatment, reflecting the residual dose from the previous day. Three punch biopsies from each participant of one formulation was obtained from the treated region during the post-treatment follow-up period between days 8 and 14 for comparison with FLIM results. RESULTS: Cellular and subcellular features associated with different epidermal and dermal layers were visualized noninvasively, down to a depth of 200 µm. Results yielded three-dimensional maps of GSK2894512 spatial distribution and residency over time. This fluorescence data provided a marker that was used as a monitor for day-to-day variance of drug presence and residency postapplication. CONCLUSIONS: The results suggest FLIM could be a viable alternative to skin biopsies without the usual patient discomfort and limitations, thereby enabling the direct measurement of skin distribution through longitudinal monitoring. These results are the first step in establishing the unique capabilities that multiphoton imaging could provide to patients through noninvasive drug detection.

Brain stem localization of neurons of the subdiaphragmatic vagus nerve fibres in the ferret (Mustela Putorius Furo): a WGA-HRP neurohistochemical study

The brain stem localization of neurons of nerve fibres in the ventral and dorsal abdominal vagal trunks were studied in the ferret. A total of 14 adult ferrets (six experimental and eight controls) were used for the study. Following anesthesia with pentobarbitone sodium, an upper midline laparotomy was done to expose the abdominal trunks of the vagus nerve. After dissecting the trunks clear of the abdominal oesophagus and the cardia of the stomach the nerve trunks were cut and WGA-HRP was applied to the proximal stump of the cut trunks. Control ferrets were divided into four groups of two ferrets. In the first group normal saline instead of the tracer was applied to the proximal stump of the vagal trunks. The second group was treated in a similar manner as the experimental animal except that the application of tracer was preceded by bilateral cervical vagotomy. In the third group of controls 0.1 ml of WGA-HRP was injected into the abdominal cavity and the fourth group had tracer injection into the hepatic portal vein. All animals were allowed a survival period of 48-72 hours after tracer injection following which each animal was perfused with normal saline, fixative and buffered sucrose. The brain stem was extracted and cut in transverse section (40 µm with the freezing microtome. Sections were then processed for WGA-HRP neurohistochemistry and subsequently viewed and analyzed under light/dark-field illuminations. In the experimental ferrets labeled cells were seen bilaterally in the dorsal motor nucleus of the vagus nerve (DMNV), the nucleus dorsomedialis (nDm), the nucleus ambiguus (nA) and the nucleus retroambiguus (nrA). The DMNV was the most intensely labeled nucleus. Sporadic distribution of labeled cells was also observed in the reticular formation (rf) between the nA and the DMNV. Labeled neurons were not seen in any of the control experiments.

Central Localisation of splenic preganglionic parsympathetic motorneurons; A wheat germ Agglutinatinin horse radish perioxidase (WGA-HRP) neurohistochemical study in the ferret (Mustela putorius furo).

Fourteen adult ferrets of both sexes and ranging in weight from 800-1500 gm were used in this investigation. Six of the ferrets were anaesthesized and a midline abdominal incision made to expose the spleen. With the aid of a Hamilton syringe and needle, the spleen of each ferret was injected with 0.1ml of 5% WGA-HRP in 0.5 M sodium chloride. The eight remaining ferrets were used as controls. Two of these had injections of 0.1ml normal saline into the spleen. The second set of two ferrets was injected with 0.1 ml of 5% WGA-HRP in buffer after bilateral trunkal vagotomy. The third set of two ferrets received intraperitoneal injection of 0.1ml of 5% WGA-HRP while in the last set the tracer was injected into the hepatic portal vein. Following the injections, the ferrets were allowed to survive for 48-72 hours after which each ferret was perfused transcardially with normal saline followed by a fixation containing paraformaldehyde and glutaraldehyde in phosphate buffer, pH 7.4 at room temperature and finally with 10% buffered sucrose at 4°C. After perfusion, the brainstem was extracted from the skull and immersed in 10% buffered sucrose and kept in the fridge overnight. Tranverse frozen sections of the brainstem were then taken, processed for WGA-HRP neurohistochemistry and analysed under light and dark field illuminations. Examination of the section taken from the six experimental ferrets revealed presence of WGA-HRP in neurons of the dorsal motor neurons in any brainstem nucleus.

Central Localisation of splenic preganglionic parsympathetic motorneurons; A wheat germ Agglutinatinin horse radish perioxidase (WGA-HRP) neurohistochemical study in the ferret (Mustela putorius furo).

Fourteen adult ferrets of both sexes and ranging in weight from 800-1500 gm were used in this investigation. Six of the ferrets were anaesthesized and a midline abdominal incision made to expose the spleen. With the aid of a Hamilton syringe and needle, the spleen of each ferret was injected with 0.1ml of 5% WGA-HRP in 0.5 M sodium chloride. The eight remaining ferrets were used as controls. Two of these had injections of 0.1ml normal saline into the spleen. The second set of two ferrets was injected with 0.1 ml of 5% WGA-HRP in buffer after bilateral trunkal vagotomy. The third set of two ferrets received intraperitoneal injection of 0.1ml of 5% WGA-HRP while in the last set the tracer was injected into the hepatic portal vein. Following the injections, the ferrets were allowed to survive for 48-72 hours after which each ferret was perfused transcardially with normal saline followed by a fixation containing paraformaldehyde and glutaraldehyde in phosphate buffer, pH 7.4 at room temperature and finally with 10% buffered sucrose at 4°C. After perfusion, the brainstem was extracted from the skull and immersed in 10% buffered sucrose and kept in the fridge overnight. Tranverse frozen sections of the brainstem were then taken, processed for WGA-HRP neurohistochemistry and analysed under light and dark field illuminations. Examination of the section taken from the six experimental ferrets revealed presence of WGA-HRP in neurons of the dorsal motor neurons in any brainstem nucleus.

BDNF and NT3 extend the critical period for developmental climbing fibre plasticity.

The effect on neonatal brain plasticity of two neurotrophins, brain derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), was studied using the rat olivocerebellar projection as a model. Unilateral transection of climbing fibres (CFs) in the rat before postnatal day 7 induces reinnervation of the deafferented hemicerebellum, but this does not occur if the transection is performed after postnatal day 10. Eleven-day-old day rats underwent unilateral CF transection followed by neurotrophin injection into the denervated cerebellar cortex 24 h later. The exogenous neurotrophins induced CF reinnervation of the denervated hemicerebellum. However BDNF was more efficacious than NT-3. Thus two neurotrophins can extend the window of neonatal brain plasticity, therefore suggesting potential therapeutic use after brain trauma.

Localization of low affinity nerve growth factor receptor in the rat inferior olivary complex during development and plasticity of climbing fibres.

The rat olivocerebellar pathway has a precise topography from an inferior olive (IOC) to Purkinje cells in the contralateral hemicerebellum. While its development and plasticity have been documented, the molecular mechanisms underlying these events are not fully elucidated. Neurotrophins are a family of growth factors with diverse roles in development and neuronal plasticity, acting through a two-receptor system, including a low affinity receptor (LNGFR) which binds all neurotrophins with similar affinity. Since neurotrophins are present in the cerebellum during early postnatal development when LNGFR is synthesized in the IOC, they may act as target-derived trophic agents for climbing fibres during development and plasticity. To assess this, standard immunohistochemistry was used to document the distribution of LNGFR in the rat IOC during climbing fibre development and until cerebellar development was complete at postnatal day 28 (P28). LNGFR immunoreactivity (LNGFR-IR) was detected in the IOC from P0 until P15, however after P7 it diminished in intensity and distribution, a change which indicates a relationship between cerebellar neurotrophins and climbing fibre development. After denervation of the left hemicerebellum, there was an apparent increase in inferior olivary LNGFR-IR that was concurrent with climbing fibre re-innervation. Thus the results of this study support the hypothesis that neurotrophins are involved in climbing fibre development and suggest a possible contribution to the plasticity of the olivocerebellar pathway.

Changes in the numbers of neurons and astrocytes during the postnatal development of the rat inferior olive.

In the developing nervous system, cell death is an important component of refining axonal projections. In the developing rat inferior olive, previous studies have demonstrated cell death as temporally incongruent with both initial axon-target interactions and subsequent axon collateral regression. Furthermore, these studies identified a late rise in neuron numbers that is concurrent with climbing fibre regression. As axonal regression has not previously been associated with increasing neuron numbers, and since immature neurons and glia have similar morphological characteristics, it was decided to reassess the timing of cell death within the inferior olive in animals in which neurons and glia had been differentially stained. Glia were identified by the presence of glial cytoskeletal proteins, S100, or glial fibrillary acidic protein, and stereological counts were made of both neurons and glia in the inferior olive from rats of ages 0, 5, 10, 15, and 30 days. The number of inferior olivary neurons was approximately 22,000 between birth and day 10, which decreased to about 17,500 by day 30 (P<0.05). In contrast, the number of glia rose from about 5,000 at birth to approximately 15,000 by day 10 (P<0.001), after which there was no further increase. The changes in neurons and glia caused the neuron-to-glia ratio to fall to approximately 1.5 by the time of functional maturation within the olive. These results confirm that there is neuronal death in the inferior olive but that it is temporally correlated with both climbing fibre regression and functional maturation of the olivocerebellar projection.

Role of afferents in the development and cell survival of the vertebrate nervous system.

1. During normal development of the vertebrate central nervous system, a considerable number of neurons die. The factors controlling which neurons die and which survive are not fully understood. 2. Target populations are known to maintain their innervating neurons. However, the role of afferents in maintaining their targets is still under review. 3. In the developing nervous system, deafferentation of a neuron population is difficult to achieve because plasticity (structural re-organization) can cause re-innervation of the area. Re-innervation alters, rather than removes, the afferent supply. 4. Afferent input is important for neuronal survival during development because deafferentation increases neuronal death by 20-30% and increasing input diminishes neuronal death. 5. Deafferented neurons die at the normal time for cell death for any given population. This occurs after the arrival of afferent axons but before the completion of connectivity and the onset of function. 6. Neuronal survival is maintained by any input, such as reinnervation by inappropriate fibres, but for optimal survival, morphological maturation and the acquisition of normal physiology, the correct input is required. 7. Afferents maintain their target neurons via a combination of electrical activity and delivery of trophic agents, which adjust intracellular calcium, thereby facilitating protein synthesis, mitochondrial function and suppressing apoptosis. 8. Evidence from animal and in vitro experiments indicates that afferents play an extremely important role in the survival of developing neurons in the immature vertebrate nervous system.

Changes in the hippocampus induced by glucose in thiamin deficient rats detected by MRI.

Using T2-weighted Magnetic Resonance Imaging (MRI) in a pyrithiamin-treated, thiamin deficient (TD) rat model of Wernicke's encephalopathy (WE), we have observed hyperintensity in the thalamus, hypothalamus, collicular bodies and hippocampus which was enhanced 40 min after a glucose load. Hyperintensity was not evident in these structures in thiamin replete rats receiving glucose nor was it enhanced in TD rats administered 2-deoxyglucose. Residual hyperintensity was still evident in the hippocampus as long as 30 days after thiamin administration and was increased by repeat glucose challenge at that time. These data indicate that the hippocampus is as vulnerable as the thalamus to some persistent pathological change when glucose is metabolised in a state of thiamin deficiency.

Acute neuronal and vascular changes following unilateral cerebellar pedunculotomy in the neonatal rat.

During development of the central nervous system (CNS) both deafferentation and axotomy induce increased neuronal death and result in a smaller brain with diminished function at maturity. Unilateral cerebellar pedunculotomy has been used as a model to study the relative importance of these 2 types of lesion on the survival of developing CNS neurons. Within the cerebellum, unilateral pedunculotomy causes deafferentation of the hemicerebellum and axotomy in the efferent pathway from the ipsilateral deep cerebellar nuclei. This results in a smaller hemicerebellum with normal cortical laminae but no extracerebellar outflow. In order to identify the sequence of events which leads to this altered structure and therefore to understand the relative importance of afferent versus target-derived trophic support, unilateral cerebellar pedunculotomy was performed on neonatal rat pups, aged between 1 and 3 days. The cerebella were analysed for histological and vascular changes after survival times of 0, 3, 6, 9, 12, 18, 21, 24 and 48 h. The results show that the effects of axotomy on the deep cerebellar nuclear neurons begin within 3 h of the lesion and apoptotic neuronal degeneration occurs within 48 h. However, the cerebellar cortical neurons continue to undergo normal histological development for at least 48 h after deafferentation. In addition, since ischaemia induces similar effects, a study of the vascular tree was made. The results indicate that the pedunculotomy does not alter the blood supply to the cerebellum, nor induce ischaemia of the cerebellar neurons. From this it may be hypothesised that target-derived trophic support is more crucial for the survival of immature neurons than is the trophic effect of afferent input.

MRI demonstration of impairment of the blood-CSF barrier by glucose administration to the thiamin-deficient rat brain.

Contrast-enhanced T1-weighted spin-echo magnetic resonance imaging (MRI) has demonstrated that Gd-diethylenetriaminepentaacetate (Gd-DTPA), which normally does not cross the blood-brain or blood-CSF barriers, does so approximately 40 min after administration of glucose to a vitamin B1 deficient rat. The period of the onset of this blood-CSF or blood-brain barrier dysfunction coincides with our previous observations of accumulation of glutamate or glutamate derivatives following an equivalent glucose load under identical conditions of thiamin deficiency, consistent with a relationship between these two observations. The dysfunction was reversed when a thiamin deficient animal was made thiamin replete.

Plasticity in the adult and neonatal central nervous system.

The adult nervous system is capable of plastic change; studies have shown that plasticity is part of normal adaptation to daily life as well as being part of the response to trauma. The structural substrates of plastic change are described, and the hypotheses for explaining functional recovery in adults following trauma are reviewed. Events in normal brain development are summarized, and experiments designed to investigate the processes involved are described. The brain of the neonate is a much more plastic structure than that of the adult, both in normal development and in response to trauma. Activity in pathways is an essential component for consolidation of connections, whether normal or compensatory. Experiments which elucidate the mechanisms of axonal/target recognition are described. Recent work on the possible development of therapeutic agents to enhance recovery from trauma, in both adults and neonates, is reviewed. An attempt is made to link the findings from basic research to the clinical field.

Cerebellar afferents in early postnatal rats: a retrograde fluorescence study.

The development of afferent projections to the cerebellum was studied by making small (20 nl) injections of True blue into the cerebella of neonatal rats under general anaesthesia. Neurones in all main precerebellar nuclei were labelled even in rats less than one day old. With the exception of the disappearance of small numbers of ipsilateral olivocerebellar projections, no qualitative differences were seen after injections in animals one or two weeks older.

Effects of partial truncal vagotomy on intragastric pressure responses to vagal stimulation and gastric distension in ferrets.

Changes in intragastric pressure after dorsal truncal vagotomy, investigated by stimulation of the surviving vagal branches and by step inflation of the stomach, were divided into an early phase lasting five days, and a late phase continuing for at least three months. During the early phase the amplitude of vagal evoked contraction was diminished but the resting pressure and the response to gastric inflation were increased. After the fifth day vagal evoked contractions doubled in amplitude but the resting pressure and the response to step inflation of the stomach returned to control levels. Ventral vagotomy did not produce any substantial changes. Alterations to gastric and body weight, or to the relation between resting pressure and evoked contraction and relaxation were excluded as causes of the enhanced vagal effectiveness. Sprouting of axons into denervated territory occurred too late to explain the changes, but an increase in synaptic density within the innervated territory has not been ruled out.

An ipsilateral olivocerebellar pathway in the normal neonatal rat demonstrated by the retrograde transport of true blue.

Neonatal rats, aged less than 24 h, were anaesthetised and 25 nl of 2% True blue was injected into the lateral part of the left cerebellar hemisphere. After 48-60 h, the brains were fixed by perfusion and 30 micron thick frozen sections were prepared of the brainstem and cerebellum. Only animals in which the injection sites were clearly unilateral were studied. In the contralateral inferior olive numerous True blue-labelled neurones were seen. In addition, the ipsilateral olive in all animals contained True blue-labelled neurones. This demonstrates that in these neonatal animals there is an ipsilateral olivocerebellar pathway.

The effect of unilateral cerebellar pedunculotomy on the vascular development of the neonatal rat cerebellum.

After cerebellar pedunculotomy the density of the blood vessel network in the cerebellar cortex was not different from that in the control animals. But the pattern of the blood vessels was different, being less organized in the operated animals.

Rate of degeneration of a neonatal ipsilateral olivocerebellar pathway revealed by unilateral cerebellar pedunculotomy in the rat.

This series of experiments tested the rate of degeneration of a recently demonstrated ipsilateral olivocerebellar pathway that followed unilateral cerebellar pedunculotomy in the neonatal rat. Animals aged either 3 or 10 days were anesthetized and subjected to a left cerebellar pedunculotomy; at the same time [3H]leucine was injected into the right inferior olive. The results indicated that there was climbing fiber labeling in the cerebellum, the extent of which depended on the age at which the experimental procedures were carried out.

Innervation of the adult rat cerebellar hemisphere by fibres from the ipsilateral inferior olive following unilateral neonatal pedunculotomy: an autoradiographic and retrograde fluorescent double-labelling study.

A left cerebellar pedunculotomy was carried out in neonatal rats of different ages to deprive the left cerebellar hemisphere of its normal climbing fibre input. In control adult animals this is totally crossed and thus arises only from the contralateral (right) inferior olive. After pedunculotomy, only the left inferior olive was intact, the right being degenerated. The remaining olivocerebellar pathway was investigated using anterograde autoradiographic or retrograde fluorescent double-labelling techniques. The anterograde autoradiographic technique showed that, in these animals, the remaining left inferior olive had an aberrant climbing fibre projection which travelled via the intact right inferior cerebellar peduncle to the denervated left hemicerebellum. If the pedunculotomy was carried out at 3 days of age (P3), this aberrant projection closely mirrored the normal pathway to the opposite hemisphere; pedunculotomy at P7 produced a different pattern of projection; while if the operation was done at P10 there was no new projection. True blue (TB) and diamidino yellow (DY) were injected into the denervated (left) and normal (right) cerebellar hemispheres respectively. Retrograde transport of these tracers confirmed both the aberrant ipsilateral projection and the normal crossed projection from neurons in the remaining inferior olive. Most of the ipsilaterally projecting neurons were in the medial accessory olive. As none of them were double-labelled, it was concluded that the new projection is not a collateral of normally projecting olivary neurons, but arises from a separate population of cells. The significance of these findings in relation to earlier work on this system is discussed.