Cuarto acto

Accurate model reproduction of targeted EAE following subarachnoid injection requires that the surgical procedure of cytokine injection causes little or no damage to the BBB, or the cingulate cortex and corpus callosum surrounding the injection site, in order to be able to attribute any subsequent pathology to the cytokines injected, rather than to the injection procedure. BBB preservation increases the relevancy of the model to pathology observed in MS cortical GMLs, which lack significant BBB disruption (Van Horssen et al., 2007).

Monastral blue tracer was confirmed to be a useful marker of the accuracy of subarachnoid injection placement, following the observation of monastral blue in the sagittal sulcus and its absence from the adjacent cortex, while substantial amounts of tracer in the cortex indicated inaccurate (intracortical) injections. Observation of the tracer beneath the dural membrane, which was removed during tissue processing, demonstrated the remarkable spread of injected material in anterior-posterior and lateral directions from the midline. Tracer studies in rats suggest that CSF flows in an anterior direction within the subarachnoid space towards the Chapter 4 – Characterisation of acute subarachnoid targeted EAE 140

cribriform plate and into the nasal lymphatics (Zhang et al., 1992). In the current study, monastral blue particles were observed to extend from the cerebellum to the olfactory bulbs but tracer appeared to be more dense in the anterior sections compared to the posterior sections at the same distance from the injection site, suggesting that diffusion of the injected cytokines occurred in both directions along the subarachnoid space, but was also influenced by the anterior direction of CSF flow. As expected, the distribution of tracer along the subarachnoid space appeared to be more limited following intracortical compared to subarachnoid injections, due to the confinement of tracer within the cortical tissue. The observation of substantial lateral spread of the tracer supports previous studies in which GMLs have been observed located on the dorsal aspect of the cortex in addition to the GMLs lining the sagittal sulcus following injection of pro-inflammatory cytokines, suggesting that diffusion of cytokines in a similar distribution pattern to that observed in the current study contributes to the pattern of GML formation (Gardner et al., 2013).

Although the glass capillaries used in the current study have a much reduced external diameter (~50µm) compared to the cited cortical injury model implements (123µm - 2mm), BBB disruption and microglial activation along the needle tract is inevitable following an intracortical injection. Indeed, microglial activation and limited demyelination was observed along the needle tract in rmMOG-immunised rats following intracortical injection of PBS in the current and previous studies (Merkler et al., 2006b; Gardner et al., 2013). In addition the more limited distribution of tracer along the subarachnoid space following intracortical injections may influence the distribution of GMLs due to altered patterns of diffusion of cytokines, planned for use in future studies. Given the wide ranging effects on the innate and adaptive immune responses which can be initiated by cortical injury and intracortical injections, animals in which the injection site was found to be intracortical rather than within the subarachnoid space were excluded from further analysis in the current and future studies.

Accurate subarachnoid placement of injections was hampered by the high degree of variation in the coronal and sagittal sutures on the skull, resulting in difficulty in precisely locating the midline. In the current study, intracortical injections were observed within 30μm of subarachnoid space, highlighting the requirement for a high degree of precision and an estimated tolerance of less than 10μm either side of the midline. Despite these factors, accurate subarachnoid injections were achieved in the majority of animals. The information yielded from these animals retains relevancy to the pathogenesis of MS, and future studies will be performed to determine the effect of raised concentrations of cytokines within the CSF and Chapter 4 – Characterisation of acute subarachnoid targeted EAE 141

specifically within a deep sulcus, with little damage to surrounding structures and preservation of the BBB (Gardner et al., 2013).

4.4 Conclusions

RmMOG batch pXVII was successfully expressed, isolated and purified from genetically modified E.coli. When titrated in vivo against a previous batch, pXIIIa, differences in cortical GM demyelinating pathology were observed following subarachnoid injection of pro- inflammatory cytokines to induce targeted EAE, which are suggested to be due to differences in epitope specificity and pathogenicity of anti-MOG antibodies between immunisation groups. We conclude that rmMOG batch pXIIIa is most suitable for subsequent immunisations. In addition, we confirmed that the sagittal sinus fills the majority of the subdural space at the dorsal surface of the sagittal sulcus and does not represent an alternative injection site, while the other sites investigated did not accurately model the enclosed space found in deep sulci where TLOs are most frequently observed in MS. We therefore conclude that injecting cytokines into the subarachnoid space at the location of the motor cortex (-0.9mm from bregma) is the most relevant model for further investigation of cytokines involved in TLO formation and the effect of meningeal inflammation on the underlying GM in vivo. In future studies, animals will be excluded from analysis if the injection is intracortical, due to the confounding variables of tissue trauma and the associated microglial activation induced by inaccurate needle placement.