Mats Hellström

Cellular tropism and transduction properties of seven adeno-associated viral vector serotypes in adult retina after intravitreal injection

Recombinant adeno-associated virus (rAAV) vectors are increasingly being used as tools for gene therapy, and clinical trials have begun in patients with genetically linked retinal disorders. Intravitreal injection is optimal for the transduction of retinal ganglion cells (RGCs), although complete selectivity has not been achieved. There may also be advantages in using intravitreal approaches for the transduction of photoreceptors. Here we compared the cellular tropism and transduction efficiency of rAAV2/1, -2/2, -2/3, -2/4, -2/5, -2/6 and -2/8 in adult rat retina after intravitreal injection. Each vector encoded green fluorescent protein (GFP), and the number, laminar distribution and morphology of transduced GFP+ cells were determined using fluorescent microscopy. Assessment of transduced cell phenotype was based on cell morphology and immunohistochemistry. rAAV2/2 and rAAV2/6 transduced the greatest number of cells, whereas rAAV2/5 and rAAV2/8 were least efficient. Most vectors primarily transduced RGCs; however, rAAV2/6 had a more diverse tropism profile, with 46% identified as amacrine or bipolar cells, 23% as RGCs and 22% as Müller cells. Müller cells were also frequently transduced by rAAV2/4. The highest photoreceptor transduction was seen after intravitreal rAAV2/3 injection. These data facilitate the design and selection of rAAV vectors to target specific retinal cells, potentially leading to an improved gene therapy for various human retinal pathologies.

Retinal ganglion cell gene therapy and visual system repair.

Recent clinical trials have shown that the use of replication deficient viral vectors to genetically modify cells in the retina can be of therapeutic benefit in the treatment of certain inherited degenerative conditions that compromise photoreceptor, and hence visual, function. This review is focussed primarily on the use of recombinant adeno-associated viral (rAAV) vectors to target neurons in inner retina, specifically retinal ganglion cells (RGCs). Genetic modification of RGCs may be of value in various ophthalmic conditions in which there is documented loss of RGCs or damage to their centrally projecting axons. Such conditions include glaucoma, optic neuritis, vascular disruption or trauma, and neurological degenerative conditions such as Alzheimers disease. Furthermore, because the retina and optic nerve (ON) form part of the CNS, the visual system is a useful experimental model in which to study the molecular and cellular mechanisms that underlie degenerative as well as regenerative responses of adult CNS neurons after injury. Gene therapy studies from a number of laboratories are first reviewed, involving not only rAAV-based treatments but also application of lentiviral and adenoviral vectors. Recent work from our own laboratory is then summarized, in which intravitreal injection of rAAV2 serotype vectors is used to introduce growth promoting genes into injured RGCs. rAAV encoding a secretable form of ciliary neurotrophic factor (CNTF) has proved to be particularly effective in promoting RGC survival and axon regeneration after optic nerve crush or after transection followed by a peripheral nerve autograft. In the latter situation we have found that RGCs and their regenerated axons are maintained for at least 15 months after the initial injury. We have also combined rAAV gene therapy with pharmacotherapy to determine if cAMP elevation and additional intravitreal injections of growth factors can act synergistically with vector-based delivery of growth-promoting genes.

Post-Injury Delivery of rAAV2-CNTF Combined with Short-Term Pharmacotherapy Is Neuroprotective and Promotes Extensive Axonal Regeneration after Optic Nerve Trauma

Recombinant adeno-associated viral (rAAV) vectors expressing neurotrophic genes reduce neuronal death and promote axonal regeneration in central nervous system (CNS) injury models. Currently, however, use of rAAV to treat clinical neurotrauma is problematic because there is a delay in the onset of transgene expression. Using the adult rat retina and optic nerve (ON), we have tested whether rAAV gene therapy administered at the time of injury combined with short-term pharmacotherapy has synergistic effects that enhance neuronal survival and regeneration. The ON was transected and a 1.5 cm segment of autologous peripheral nerve (PN) was grafted onto the cut end. At this time, bicistronic rAAV2 encoding ciliary neurotrophic factor (CNTF) and green fluorescent protein (rAAV2-CNTF-GFP) was injected into the injured eye. To provide interim support for axotomized retinal ganglion cells (RGCs) during vector integration and therapeutic transgene expression, rCNTF protein and a cyclic adenosine monophosphate (cAMP) analogue (CPT-cAMP) were injected intravitreally 3 and 10 days postoperatively. For comparison, another rAAV2-CNTF-GFP group received two intravitreal saline injections 3 and 10 days after the PN-ON surgery. A further PN graft group received only postoperative intravitreal injections of rCNTF plus CPT-cAMP. After 4 weeks, regenerating RGCs were retrogradely labelled by applying fluorogold to the distal end of each PN graft. Compared to saline-injected animals, both RGC survival and axonal regrowth were significantly higher in the rCNTF and CPT-cAMP injected rAAV2-CNTF-GFP group; approximately one third of the RGC population survived axotomy, and 27% of these regrew an axon. These values were also higher than those obtained in rats that received only rCNTF plus CPT-cAMP injections. Therefore, we show for the first time that rAAV-mediated gene delivery at the time of, or just after, neurotrauma is most successful when combined with temporary post-injury trophic support, and is potentially a viable treatment strategy for patients after acute CNS injury.

Inhibitory effect of punicalagin on lipopolysaccharide-induced neuroinflammation, oxidative stress and memory impairment via inhibition of nuclear factor-kappaB.

&NA; Neuroinflammation is significant in the pathogenesis and development of Alzheimers disease (AD). Previously, we showed lipopolysaccharide (LPS)‐induced neuroinflammation caused memory impairment. We investigated the possible preventive effects of punicalagin (PUN), a component of pomegranate, on memory deficiency caused by LPS, along with the fundamental mechanisms. LPS‐treated cultured astrocytes and microglial BV‐2 cells were investigated for anti‐neuroinflammatory effects of PUN. PUN (1.5 mg/kg) ameliorates LPS (250 &mgr;g/kg daily 7 times)‐induced memory impairment as well as prevents the LPS‐induced expression of inflammatory proteins. In in vitro study, we also found that PUN (1 &mgr;g/ml) inhibited the LPS‐(10, 20 and 50 &mgr;M) induced expression of iNOS and Cox‐2 as well as the production of ROS, NO, TNF‐&agr; and IL‐1&bgr;. PUN also suppress activation of NF‐&kgr;B via inhibition of I&kgr;B degradation as well as p50 and p65 translocation into the nucleus in LPS treated mouse brain and cultured astrocytes and microglial BV‐2 cells. Consistent with the inhibitory effect on neuro inflammation, PUN inhibited LPS‐induced A&bgr;1‐42 generation through down‐regulation of APP and BACE1 expression in in vivo and in vitro study. Moreover, PUN directly binds to NF‐&kgr;B subunit p50 evidenced by a docking model and pull down assay. These results suggest that PUN inhibits LPS‐induced memory impairment via anti‐inflammatory and anti‐amylogenic mechanisms through inhibition of NF‐&kgr;B activation. HighlightsNeuroinflammation and amyloidogenesis are main symptoms of Alzheimers disease.NF‐&kgr;B activation can induce the inflammation and amyloidogenesis pathways.Punicalagin inhibits NF‐&kgr;B activation through direct binding to its subunit P50.Punicalagin reduces LPS‐induced neuroinflammation and amyloidogenesis.Punicalagin is a possible candidate for treating Alzheimers disease.

Luminal solutions protect mucosal barrier during extended preservation

BACKGROUND Mucosal barrier injury during intestinal preservation (IP) and transplantation favors life-threatening infections. Luminal delivery of solutions containing amino acids or polyethylene glycols (PEGs) may improve preservation results and reduce this injury. We tested if solutions containing glutamine and PEG influence the mucosal injury. MATERIALS AND METHODS Rat intestines were perfused and stored in Viaspan-University of Wisconsin solution. Before IP, a PEG 3350 solution was introduced intraluminally alone (group 1) or supplemented with 40 mmol/L L-glutamine (group 2). Controls underwent vascular flush alone (group 3). Preservation injury was evaluated after 8, 14, and 24 h by histology and goblet cell count. Tight-junction proteins zonula occludens-1, claudin-3, claudin-4, and caveolin-1 were studied by immunofluorescence. Maltase and caspase-3 activity were also analyzed. RESULTS Group 1 showed mild edema at 8 h and mucosal disruption by 24 h; these features were greatly improved in group 2 where continuous mucosa was found after 24 h of IP. Intestines in group 3 did worse at all time points with subepithelial edema (Park/Chiu grade 3) and marked goblet cell depletion; caspase-3 activity was lowest in group 2. Tight-junction proteins varied continuously during IP; zonula occludens-1 expression and colocalization with claudins decreased significantly in group 3 but not in other groups. Claudin-3 was distinctly localized in the membrane, but stained diffuse, cytoplasmic at later time-points. Claudin-4 changed to a cytoplasmic granular pattern. No caveolin-1 colocalization was observed. CONCLUSIONS Luminal PEG and glutamine delay epithelial breakdown and preserve several important mucosal features during extended IP.

Large-scale reconstitution of a retina-to-brain pathway in adult rats using gene therapy and bridging grafts: An anatomical and behavioral analysis.

Peripheral nerve (PN) grafts can be used to bridge tissue defects in the CNS. Using a PN-to-optic nerve (ON) graft model, we combined gene therapy with pharmacotherapy to promote the long-distance regeneration of injured adult retinal ganglion cells (RGCs). Autologous sciatic nerve was sutured onto the transected ON and the distal end immediately inserted into contralateral superior colliculus (SC). Control rats received intraocular injections of saline or adeno-associated virus (AAV) encoding GFP. In experimental groups, three bi-cistronic AAV vectors encoding ciliary neurotrophic factor (CNTF) were injected into different regions of the grafted eye. Each vector encoded a different fluorescent reporter to assess retinotopic order in the regenerate projection. To encourage sprouting/synaptogenesis, after 6 weeks some AAV-CNTF injected rats received an intravitreal injection of recombinant brain-derived neurotrophic factor (rBDNF) or AAV-BDNF. Four months after surgery, cholera toxin B was used to visualize regenerate RGC axons. RGC viability and axonal regrowth into SC were significantly greater in AAV-CNTF groups. In some cases, near the insertion site, regenerate axonal density resembled retinal terminal densities seen in normal SC. Complex arbors were seen in superficial but not deep SC layers and many terminals were immunopositive for presynaptic proteins vGlut2 and SV2. There was improvement in visual function via the grafted eye with significantly greater pupillary constriction in both AAV-CNTF+BDNF groups. In both control and AAV-CNTF+rBDNF groups the extent of light avoidance correlated with the maximal distance of axonal penetration into superficial SC. Despite the robust regrowth of RGC axons back into the SC, axons originating from different parts of the retina were intermixed at the PN graft/host SC interface, indicating that there remained a lack of order in this extensive regenerate projection.

Parkin deficiency exacerbate ethanol-induced dopaminergic neurodegeneration by P38 pathway dependent inhibition of autophagy and mitochondrial function

Parkinsons disease (PD) is a neurodegenerative disease characterized by selective degeneration of dopaminergic neurons in the substantia nigra. Parkin (which encoded by Park2), an E3 ubiquitin ligase, is the most frequently mutated gene that has casually been linked to autosomal recessive early onset familial PD. We tested the effect of Park2 on ethanol-induced dopaminergic neurodegeneration in Park2 knockout (KO) transgenic mice after chronic ethanol feeding. Male Park2 wild type (WT) and KO mice (8 weeks old) were fed on a Lieber-DeCarli diet containing 6.6% ethanol for 2 weeks, and compared their responses. We found that knockout of Park2 exacerbates ethanol-induced behavioral impairment as well as dopamine depletion. In the mechanism study, we found that knockout of Park2 increased reactive oxygen species (ROS) production, mitophagy formation, mitochondrial dysfunction, and expression of pro-apoptotic proteins, but decreased expression of pro-autophagic proteins. Knockout of Park2 also increased ethanol-induced activation of p38 mitogen-activated protein kinase. In addition, ROS production, mitophagy formation, mitochondrial dysfunction, and expression of pro-apoptotic proteins were increased, but expression of pro-autophagic proteins were decreased by a treatment of ethanol (100 μM) in Park2 siRNA-transfacted PC12 cells (5 μM). Moreover, the exacerbating effects of Park2 deletion on ethanol-induced ROS generation, mitophagy, mitochondrial dysfunction as well as cell death were reduced by p38 specific inhibitor (SB203580) in in vitro (10 μM) and in vivo 10 mg/kg). Park2 deficiency exacerbates ethanol-induced dopaminergic neuron damage through p38 kinase dependent inhibition of autophagy and mitochondrial function.

Anti-Inflammatory Effect of Titrated Extract of Centella asiatica in Phthalic Anhydride-Induced Allergic Dermatitis Animal Model

Centella asiatica has potent antioxidant and anti-inflammatory properties. However, its anti-dermatitic effect has not yet been reported. In this study, we investigated the anti-dermatitic effects of titrated extract of Centella asiatica (TECA) in a phthalic anhydride (PA)-induced atopic dermatitis (AD) animal model as well as in vitro model. An AD-like lesion was induced by the topical application of five percent PA to the dorsal skin or ear of Hos:HR-1 mouse. After AD induction, 100 μL of 0.2% and 0.4% of TECA (40 μg or 80 μg/cm2) was spread on the dorsum of the ear or back skin three times a week for four weeks. We evaluated dermatitis severity, histopathological changes and changes in protein expression by Western blotting for inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and NF-κB activity, which were determined by electromobility shift assay (EMSA). We also measured TNF-α, IL-1β, IL-6, and IgE concentration in the blood of AD mice by enzyme-linked immunosorbent assay (ELISA). TECA treatment attenuated the development of PA-induced atopic dermatitis. Histological analysis showed that TECA inhibited hyperkeratosis, mast cells and infiltration of inflammatory cells. TECA treatment inhibited expression of iNOS and COX-2, and NF-κB activity as well as the release of TNF-α, IL-1β, IL-6, and IgE. In addition, TECA (1, 2, 5 μg/mL) potently inhibited Lipopolysaccharide (LPS) (1 μg/mL)-induced NO production, expression of iNOS and COX-2, and NF-κB DNA binding activities in RAW264.7 macrophage cells. Our data demonstrated that TECA could be a promising agent for AD by inhibition of NF-κB signaling.

The microsurgical training programme in Gothenburg, Sweden: early experiences

Abstract Objective: Microsurgical techniques are increasingly used in routine surgical practice as well as in biomedical research. The training opportunities at standardised training courses are limited, and no microsurgical training facility or programme existed in Scandinavia before 2013. Methods: A microsurgery laboratory was set up and two different courses were started, aiming separately at biomedical researchers and surgeons. The course for biomedical researchers teaches basic microsurgical skills such as vessel isolation, cannulation, and arterial microvascular suture under magnification. The more advanced course for surgeons focuses on various techniques of microvascular and nerve anastomosis. Both courses use a combination of theory and practice, with emphasis on the practical part, the course for surgeons also includes clinically relevant information. Results: Twelve 5-day courses using both non-living models and exercises on laboratory animals have been conducted and attended by 49 researchers and 44 surgeons. The organisation and the programme of the training courses as well as ‘The 4E concept’ behind the course (educational curriculum, equipment, ergonomy, and evaluation) are further detailed. Conclusions: We have successfully established the first training laboratory and series of microsurgical training courses in Scandinavia at two different levels. The experience from the first 12 courses shows the need for this type of structured training, and confirms that the microsurgical education curriculums needs to be adapted to participants’ prerequisites and expectations, and various difficulty levels should be considered.

Luminal Polyethylene Glycol Alleviates Intestinal Preservation Injury Irrespective of Molecular Size

Intestinal preservation injury (IPI) and the resulting mucosa injury raise several serious challenges early after intestinal transplantation. The current clinical approach using only vascular perfusion allows the shortest preservation period among the abdominal organs. The experimental addition of luminal polyethylene glycol (PEG) solutions has been repeatedly suggested to alleviate preservation injury, improve graft quality, and prolong the preservation time. We investigated whether the molecular mass of PEG in solution influences the development of intestinal preservation injury. Small intestines of Sprague-Dawley rats were perfused with University of Wisconsin solution. Group 1 underwent vascular perfusion only (clinical control), group 2 received additional luminal PEG3350 Da, group 3 received luminal PEG10000 Da, and group 4 received luminal PEG20000 Da (n = 8/group). Tissue samples were obtained after 4, 8, and 14 hours. We studied the tissue damage (Chiu/Park score, Goblet cells, apoptosis, tight junctions), activation of c-Jun NH2-terminal kinase (JNK), and p38-mitogen-activated protein kinase (MAPK), and we performed Ussing chamber assessments. Mucosal morphologic and electrophysiologic parameters were significantly improved in the groups receiving luminal PEG. There was significantly less apoptotic activity in groups 2, 3, and 4. Both MAPKs revealed an activation peak after 4 hours with group 3 showing lesser p38-MAPK activation. PEG 20 kDa interfered with protein immunodetection. The results indicate that luminal solutions of PEG of medium and large molecular mass significantly delay the onset and development of IPI, providing further evidence that luminal interventions may allow for longer cold storage intervals of intestinal grafts.