BackgroundCalreticulin (CRT) is a chaperone protein, which aids correct folding of glycosylated proteins in the endoplasmic reticulum (ER). Under conditions of ER stress, CRT is upregulated and may be displayed on the surface of cells or be secreted. This ‘ecto-CRT’ may activate the innate immune response or it may aid clearance of apoptotic cells. Our and other studies have demonstrated upregulation of ER stress markers CHOP, BiP, ATF4, XBP1 and phosphorylated e-IF2 alpha (p-eIF2 alpha) in biopsy and post-mortem human multiple sclerosis (MS) samples. We extend this work by analysing changes in expression of CRT, BiP, CHOP, XBP1 and p-eIF2 alpha in a rat model of inflammatory demyelination. Demyelination was induced in the spinal cord by intradermal injection of recombinant mouse MOG mixed with incomplete Freund’s adjuvant (IFA) at the base of the tail. Tissue samples were analysed by semi-quantitative scoring of immunohistochemically stained frozen tissue sections. Data generated following sampling of tissue from animals with spinal cord lesions, was compared to that obtained using tissue derived from IFA- or saline-injected controls. CRT present in rat serum and in a cohort of human serum derived from 14 multiple sclerosis patients and 11 healthy controls was measured by ELISA.ResultsStained tissue scores revealed significantly (p<0.05) increased amounts of CRT, CHOP and p-eIF2 alpha in the lesion, lesion edge and normal-appearing white matter when compared to controls. CHOP and p-eIF2 alpha were also significantly raised in regions of grey matter and the central canal (p<0.05). Immunofluorescent dual-label staining confirmed expression of these markers in astrocytes, microglia or neurons. Dual staining of rat and human spinal cord lesions with Oil Red O and CRT antibody showed co-localisation of CRT with the rim of myelin fragments. ELISA testing of sera from control and EAE rats demonstrated significant down-regulation (p<0.05) of CRT in the serum of EAE animals, compared to saline and IFA controls. This contrasted with significantly increased amounts of CRT detected in the sera of MS patients (p<0.05), compared to controls.ConclusionThis data highlights the potential importance of CRT and other ER stress proteins in inflammatory demyelination.

Treatment of neurodegenerative disease is entering a new era where direct intracerebral delivery of therapeutic factors aims to restore normality to dysfunctional circuits.Cell-based therapeutic approaches, where virally manipulated mesenchymal stem cells (MSCs) over-expressing glial cell line derived neurotrophic factor (GDNF) are utilised as vehicles to deliver neurotrophic support to the Parkinsonian brain, have shown promising pre-clinical results at preserving dopaminergic neuron integrity. However, poor cell survival following transplantation will hinder clinical progression. One approach to improve MSCs survival following transplantation is to couple the cell engraftment procedure with a scaffold thereby providing a physical substrate upon which to eventually complex pro-survival factors. Evaluation of commercially available, clinically accepted materials with an established safety profile will expedite clinical translation. Therefore, this study sought to determine if a clinically used fibrin scaffold can be utilised as an adjunct to intra-cerebral cell transplantation without evoking an adverse host or stem cell response. Sixteen male SpragueDawley rats received bilateral intra-striatal transplants of 30,000 GDNF-transduced MSCs delivered in either control transplantation medium or a fibrin scaffold. Rats were sacrificed 1, 4, 7, and 14 days post-transplantation. Brains were analysed to determine in situ polymerisation and biodegradability of the fibrin scaffold, GDNF release from transplanted GDNF-MSCs, survival of the GDNF-MSCs graft and the host's immune response to the transplant. This study found that fibrin scaffold was adaptable to intra-cerebral delivery with successful polymerisation of the fibrin scaffold in situ. Inclusion of the fibrin scaffold was not detrimental to cell survival nor did it impede neurotrophin release from entrapped cells.Importantly, the inclusion of the fibrin scaffold was associated with a reduced host astroglial and microglial response compared to cells alone indicative of a favourable biocompatibility profile. Overall, fibrin represents an adaptable scaffold for inclusion in a minimally invasive cell-based therapeutic approach for neurodegenerative diseases.TEXT Introduction: