Emma Castrique

Corticosteroids mediate fast feedback of the rat hypothalamic-pituitary-adrenal axis via the mineralocorticoid receptor.

The aim of this study was to investigate fast corticosteroid feedback of the hypothalamic-pituitary-adrenal (HPA) axis under basal conditions, in particular the role of the mineralocorticoid receptor. Blood samples were collected every 5 min from conscious rats at the diurnal peak, using an automated blood sampling system, and assayed for corticosterone. Feedback inhibition by rapidly increasing concentrations of ligand was achieved with an intravenous bolus of exogenous corticosteroid. This resulted in a significant reduction in plasma corticosterone concentrations within 23 min of the aldosterone bolus and 28 min of methylprednisolone. Evaluation of the pulsatile secretion of corticosterone revealed that the secretory event in progress at the time of administration of exogenous steroid was unaffected, whereas the next secretory event was inhibited by both aldosterone and methylprednisolone. The inhibitory effect of aldosterone was limited in duration (1 secretory event only), whereas that of methylprednisolone persisted for 4-5 h. Intravenous administration of canrenoate (a mineralocorticoid receptor antagonist) also had rapid effects on the HPA axis, with an elevation of ACTH within 10 min and corticosterone within 20 min. The inhibitory effect of aldosterone was unaffected by pretreatment with the glucocorticoid receptor antagonist RU-38486 but blocked by the canrenoate. These data imply an important role for the mineralocorticoid receptor in fast feedback of basal HPA activity and suggest that mineralocorticoids can dynamically regulate basal corticosterone concentrations during the diurnal peak, a time of day when there is already a high level of occupancy of the cytoplasmic mineralocorticoid receptor.

Regulation of the Hypothalamic-Pituitary-Adrenal Axis Circadian Rhythm by Endocannabinoids Is Sexually Diergic

We have examined the effects of acute administration of the cannabinoid receptor type 1 (CB1) antagonist AM251 on the rat hypothalamic-pituitary-adrenal (HPA) axis with respect to both gender and time of day. Blood samples were collected from conscious male and female rats every 5 min using an automated blood sampling system, and corticosterone concentrations were determined. In male rats, there was a distinct diurnal effect of AM251 with a greater activation of the HPA axis in the morning (diurnal trough) compared with the evening (diurnal peak). At both times of the day, circulating corticosterone concentrations were elevated for approximately 4 h after AM251 administration. In female rats, there was also diurnal variation in the activation of the HPA axis; however, these effects were not as profound as those in males. Corticosterone concentrations were only slightly elevated at the diurnal trough and for a shorter time period than in males (2 compared with 4 h). Moreover, there was no effect of AM251 on corticosterone concentrations when administered at the diurnal peak. Subsequent studies, only in males, in which both ACTH and corticosterone were measured, confirmed that the effects of AM251 on corticosterone were mediated by ACTH. Moreover, the elevation of both ACTH and corticosterone could be replicated using another CB1 antagonist, AM281. These data demonstrate that the extent and duration of HPA axis activation after CB1 blockade are clearly dependent on both gender and time of day.

Use of a prolactin-Cre/ROSA-YFP transgenic mouse provides no evidence for lactotroph transdifferentiation after weaning, or increase in lactotroph/somatotroph proportion in lactation

In rats, a shift from somatotroph dominance to lactotroph dominance during pregnancy and lactation is well reported. Somatotroph to lactotroph transdifferentiation and increased lactotroph mitotic activity are believed to account for this and associated pituitary hypertrophy. A combination of cell death and transdifferentiation away from the lactotroph phenotype has been reported to restore non-pregnant pituitary proportions after weaning. To attempt to confirm that a similar process occurs in mice, we generated and used a transgenic reporter mouse model (prolactin (PRL)-Cre/ROSA26-expression of yellow fluorescent protein (EYFP)) in which PRL promoter activity at any time resulted in permanent, stable, and highly specific EYFP. Triple immunochemistry for GH, PRL, and EYFP was used to quantify EYFP+ve, PRL−ve, and GH+ve cell populations during pregnancy and lactation, and for up to 3 weeks after weaning, and concurrent changes in cell size were estimated. At all stages, the EYFP reporter was expressed in 80% of the lactotrophs, but in fewer than 1% of other pituitary cell types, indicating that transdifferentiation from those lactotrophs where reporter expression was activated is extremely rare. Contrary to expectations, no increase in the lactotroph/somatotroph ratio was seen during pregnancy and lactation, whether assessed by immunochemistry for the reporter or PRL: findings confirmed by PRL immunochemistry in non-transgenic mice. Mammosomatotrophs were rarely encountered at the age group studied. Individual EYFP+ve cell volumes increased significantly by mid-lactation compared with virgin animals. This, in combination with a modest and non-cell type-specific estrogen-induced increase in mitotic activity, could account for pregnancy-induced changes in overall pituitary size.

Intrastriatal convection-enhanced delivery results in widespread perivascular distribution in a pre-clinical model

BackgroundConvection-enhanced delivery (CED), a direct method for drug delivery to the brain through intraparenchymal microcatheters, is a promising strategy for intracerebral pharmacological therapy. By establishing a pressure gradient at the tip of the catheter, drugs can be delivered in uniform concentration throughout a large volume of interstitial fluid. However, the variables affecting perivascular distribution of drugs delivered by CED are not fully understood. The aim of this study was to determine whether the perivascular distribution of solutes delivered by CED into the striatum of rats is affected by the molecular weight of the infused agent, by co-infusion of vasodilator, alteration of infusion rates or use of a ramping regime. We also wanted to make a preliminary comparison of the distribution of solutes with that of nanoparticles.MethodsWe analysed the perivascular distribution of 4, 10, 20, 70, 150 kDa fluorescein-labelled dextran and fluorescent nanoparticles at 10 min and 3 h following CED into rat striatum. We investigated the effect of local vasodilatation, slow infusion rates and ramping on the perivascular distribution of solutes. Co-localisation with perivascular basement membranes and vascular endothelial cells was identified by immunohistochemistry. The uptake of infusates by perivascular macrophages was quantified using stereological methods.ResultsWidespread perivascular distribution and macrophage uptake of fluorescein-labelled dextran was visible 10 min after cessation of CED irrespective of molecular weight. However, a significantly higher proportion of perivascular macrophages had taken up 4, 10 and 20 kDa fluorescein-labelled dextran than 150 kDa dextran (p < 0.05, ANOVA). Co-infusion with vasodilator, slow infusion rates and use of a ramping regime did not alter the perivascular distribution. CED of fluorescent nanoparticles indicated that particles co-localise with perivascular basement membranes throughout the striatum but, unlike soluble dextrans, are not taken up by perivascular macrophages after 3 h.ConclusionsThis study suggests that widespread perivascular distribution and interaction with perivascular macrophages is likely to be an inevitable consequence of CED of solutes. The potential consequences of perivascular distribution of therapeutic agents, and in particular cytotoxic chemotherapies, delivered by CED must be carefully considered to ensure safe and effective translation to clinical trials.

The development of an implantable catheter system for chronic or intermittent convection-enhanced delivery

Convection-enhanced delivery (CED) is a promising technique for the administration of therapeutic agents such as cytotoxics, neurotrophins and enzymes to the brain. In this study we describe the development of an implantable catheter system that is compatible with long-term intermittent CED. Catheters made from fused silica, PEEK or carbothane, and of various internal and external diameters were implanted in the striatum of rats and assessed for patency at 21 or 28 days. A high-rate of catheter blockage was observed with all fused silica and PEEK catheters. Carbothane catheters with an outer diameter of 0.6mm and an inner diameter of 0.35 mm had significantly lower rates of blockage (P≤0.01). Carbothane catheters were then implanted into 4 Large White/Landrace pigs and 4 NIH miniature pigs and infusions undertaken at monthly intervals to evaluate catheter patency and infusate distribution. Catheter patency was demonstrated for a maximum period of 163 days in one animal. Widespread and reproducible intraputamenal CED could be achieved with intermittent drug delivery at flow-rates as high as 5 μl/min. Problems were encountered using the pig model due to catheter distortion from rapid animal growth. In conclusion, it is possible to achieve intermittent high-flow CED with a chronic implanted carbothane catheter with a low rate of catheter blockage.

Evaluation and Optimization of the Administration of Recombinant Adeno-Associated Viral Vectors (Serotypes 2/1, 2/2, 2/rh8, 2/9, and 2/rh10) by Convection-Enhanced Delivery to the Striatum

Convection-enhanced delivery (CED) of recombinant adeno-associated virus (rAAV) vectors is a promising approach for delivery of therapeutic transgenes to the brain. In this study we have systematically examined vector dosing in vivo. Infusions of rAAV serotypes 2/1, 2/2, 2/rh8, 2/9, and 2/rh10 expressing an enhanced green fluorescent protein reporter gene were undertaken into the striatum of rats and pigs using CED. Vector distribution, as defined by the volume of distribution and number of transduced cells following each infusion, was determined using stereological methods. Immunohistochemistry was used to determine the transductional tropism of serotypes and to evaluate for the presence of immune cell infiltration into the brain. Vector distribution was highly variable between serotypes. Infusion rate had no significant effect on vector distribution or the occurrence of tissue damage. For serotypes 2/1, 2/2 and 2/rh10, as the vector concentration was increased beyond 10(12) vg/ml, no increase in vector distribution was observed. In contrast, for serotypes 2/rh8 and 2/9, retrograde axonal transport was observed above this threshold concentration. Cell transduction was principally neuronal for all serotypes and was associated with a low-level immune response. In planning clinical trials it is critical that these observations are considered in order to achieve optimal vector dosing.

Clearance and Toxicity of Recombinant Methionyl Human Glial Cell Line-Derived Neurotrophic Factor (r-metHu GDNF) Following Acute Convection-Enhanced Delivery into the Striatum

Background Despite promising early results, clinical trials involving the continuous delivery of recombinant methionyl human glial cell line-derived neurotrophic factor (r-metHuGDNF) into the putamen for the treatment of Parkinsons disease have shown evidence of poor distribution and toxicity due to point-source accumulation. Convection-enhanced delivery (CED) has the potential to facilitate more widespread and clinically effective drug distribution. Aims We investigated acute CED of r-metHuGDNF into the striatum of normal rats in order to assess tissue clearance, toxicity (neuron loss, gliosis, microglial activation, and decreases in synaptophysin), synaptogenesis and neurite-outgrowth. We investigated a range of clinically relevant infused concentrations (0.1, 0.2, 0.6 and 1.0 µg/µL) and time points (2 and 4 weeks) in order to rationalise a dosing regimen suitable for clinical translation. Results Two weeks after single dose CED, r-metHuGDNF was below the limit of detection by ELISA but detectable by immunohistochemistry when infused at low concentrations (0.1 and 0.2 µg/µL). At these concentrations, there was no associated neuronal loss (neuronal nuclei, NeuN, immunohistochemistry) or synaptic toxicity (synaptophysin ELISA). CED at an infused concentration of 0.2 µg/µL was associated with a significant increase in synaptogenesis (p<0.01). In contrast, high concentrations of r-metHuGDNF (above 0.6 µg/µL) were associated with neuronal and synaptic toxicity (p<0.01). Markers for gliosis (glial fibrillary acidic protein, GFAP) and microglia (ionized calcium-binding adapter molecule 1, Iba1) were restricted to the needle track and the presence of microglia had diminished by 4 weeks post-infusion. No change in neurite outgrowth (Growth associated protein 43, GAP43, mRNA) compared to artificial cerebral spinal fluid (aCSF) control was observed with any infused concentration. Conclusion The results of this study suggest that acute CED of low concentrations of GDNF, with dosing intervals determined by tissue clearance, has most potential for effective clinical translation by optimising distribution and minimising the risk of toxic accumulation.

Convection-enhanced delivery of AAV2 in white matter—A novel method for gene delivery to cerebral cortex

BACKGROUND Convection-enhanced delivery (CED) is currently under investigation for delivering therapeutic agents to subcortical targets in the brain. Direct delivery of therapies to the cerebral cortex, however, remains a significant challenge. NEW METHOD We describe a novel method of targeting adeno-associated viral vector (AAV) mediated gene therapies to specific cerebral cortical regions by performing high volume, high flow rate infusions into underlying white matter in a large animal (porcine) model. RESULTS Infusion volumes of up to 700 μl at flow rates as high as 10 μl/min were successfully performed in white matter without adverse neurological sequelae. Co-infusion of AAV2/5-GFP with 0.2% Gadolinium in artificial CSF confirmed transgene expression in the deep layers of cerebral cortex overlying the infused areas of white matter. COMPARISON WITH EXISTING METHODS AAV-mediated gene therapies have been previously targeted to the cerebral cortex by performing intrathalamic CED and exploiting axonal transport. The novel method described in this study facilitates delivery of gene therapies to specific regions of the cerebral cortex without targeting deep brain structures. CONCLUSIONS AAV-mediated gene therapies can be targeted to specific cortical regions by performing CED into underlying white matter. This technique could be applied to the treatment of neurological disorders characterised by cerebral cortical degeneration.

An evaluation of site-specific immune responses directed against first-generation adenoviral vectors administered by convection-enhanced delivery.

Direct adenoviral vector injections into the brain have been used in clinical trials to treat patients with high‐grade gliomas. However, a recent phase 3 trial using first‐generation vectors failed to demonstrate significant survival benefits. Malignant gliomas infiltrate extensively through the white matter, making them difficult to treat, and chemotherapy is at best partially effective. Convection enhanced delivery (CED) represents a rationale approach for achieving widespread targeting of infiltrating tumour cells. Previous studies have demonstrated that infusions of particle numbers above a threshold level [108 plaque‐forming units (pfu)] are associated with a pronounced inflammatory response in rat grey matter, although no such comparisons have been made with CED infusions into the white matter.