Anna-Karin Persson

Susceptibility to chronic pain following nerve injury is genetically affected by CACNG2

Chronic neuropathic pain is affected by specifics of the precipitating neural pathology, psychosocial factors, and by genetic predisposition. Little is known about the identity of predisposing genes. Using an integrative approach, we discovered that CACNG2 significantly affects susceptibility to chronic pain following nerve injury. CACNG2 encodes for stargazin, a protein intimately involved in the trafficking of glutamatergic AMPA receptors. The protein might also be a Ca(2+) channel subunit. CACNG2 has previously been implicated in epilepsy. Initially, using two fine-mapping strategies in a mouse model (recombinant progeny testing [RPT] and recombinant inbred segregation test [RIST]), we mapped a pain-related quantitative trait locus (QTL) (Pain1) into a 4.2-Mb interval on chromosome 15. This interval includes 155 genes. Subsequently, bioinformatics and whole-genome microarray expression analysis were used to narrow the list of candidates and ultimately to pinpoint Cacng2 as a likely candidate. Analysis of stargazer mice, a Cacng2 hypomorphic mutant, provided electrophysiological and behavioral evidence for the genes functional role in pain processing. Finally, we showed that human CACNG2 polymorphisms are associated with chronic pain in a cohort of cancer patients who underwent breast surgery. Our findings provide novel information on the genetic basis of neuropathic pain and new insights into pain physiology that may ultimately enable better treatments.

Fecal transplant against relapsing Clostridium difficile-associated diarrhea in 32 patients

Abstract Clostridium difficile-associated disease (CDAD) with frequent watery stools, sometimes with painful bowel movements, fever and sickness, is probably the major known cause of antibiotic-associated diarrhea and colitis, most probably depending on a disruption of the normal intestinal balance in the microbiome. In this study, we have inoculated a mixture of fecal microbes – as an enema – originating from a healthy Scandinavian middle-aged donor, regularly re-cultivated under strict anaerobic conditions for more than 10 years, to 32 patients. Twenty-two patients (69%) were durably cured. In those patients receiving the transplant by colonoscopy, four out of five were cured. To the best of our knowledge, this is the first time a fecal culture of microbes has retained the possibility for years to cure a substantial number of patients with CDAD.

Behavioral changes and trigeminal ganglion sodium channel regulation in an orofacial neuropathic pain model

Abstract We used a photochemical method to generate a partial ischemic injury to the infraorbital branch of the trigeminal nerve in rats. Following injury, rats developed a bilateral persistent hypersensitivity to mechanical stimulation in the territory innervated by the infraorbital nerve. In addition, spread of mechanical hypersensitivity beyond the facial region was noted. Heat hypersensitivity was also present, although to a lesser extent and of a shorter duration. In some rats, excessive facial grooming/scratching were observed. Morphological examination revealed a graded damage to the irradiated portion of the infraorbital nerve that was related to the duration of laser irradiation. Investigations of gene expression changes in injured trigeminal ganglion neurons of animals with behavioral signs of neuropathic pain demonstrated that the sodium channel α‐subunit Nav1.3—absent in sham‐operated animals—was expressed to a limited extent. mRNAs for Nav1.8 and Nav1.9 were reduced both with respect to proportions of expressing neurons and to intensities, whereas the β3 subunit was markedly upregulated. mRNA levels of p11, a regulatory factor that facilitates the surface expression of Nav1.8, were unchanged. Previous findings have shown that injury to the trigeminal nerve branches may elicit responses that differ from those of segmental spinal nerves. Despite this we conclude that the key sodium channel regulations that are reported as consequences of nerve damage in the dorsal root ganglia seem to appear also in the trigeminal ganglion. Thus, novel analgesic drugs designed to target the sodium channel subtypes involved could be of use for the treatment of orofacial pain.

Autotomy behavior correlates with the DRG and spinal expression of sodium channels in inbred mouse strains.

Patients who have suffered nerve injury show profound inter-individual variability in neuropathic pain even when the precipitating injury is nearly identical. Variability in pain behavior is also observed across inbred strains of mice where it has been attributed to genetic polymorphisms. Identification of cellular correlates of pain variability across strains can advance the understanding of underlying pain mechanisms. Voltage-gated sodium channels (VGSCs) play a major role in the generation and propagation of action potentials in the primary afferents and are therefore of obvious importance for pain phenotype. Here, we examined the mRNA expression levels of the VGSC alpha-subunits Na(v)1.3, Na(v)1.5, Na(v)1.6, and Na(v)1.7, as well as the auxiliary VGSC-related molecule, Contactin. Dorsal root ganglia (DRG) and spinal cords from 5 inbred mouse strains with contrasting pain phenotype (AKR/J, C3H/HeJ, C57BL/6J, C58/J and CBA/J) were analyzed 7 days following sciatic and saphenous nerve transection. In the DRG, Na(v)1.6, Na(v)1.7 and Contactin were abundantly expressed in control animals. Following nerve injury, the residual mRNA levels of Na(v)1.6 (downregulated in two of the strains) correlated tightly to the extent of autotomy behavior. A suggestive correlation was also seen for the post-injury mRNA levels of Contactin (downregulated in all strains) with autotomy. Thus, our results suggest a contribution by DRG Na(v)1.6, and possibly Contactin to neuropathic pain in the neuroma model in mice.

Expression of DRG candidate pain molecules after nerve injury--a comparative study among five inbred mouse strains with contrasting pain phenotypes.

Neuropathic pain that develops after trauma to a nerve may be caused by altered transcription of genes in the damaged neurons. We have previously investigated the effect of nerve injury on the expression of six dorsal root ganglion (DRG) pain candidate molecules in five inbred mouse strains with different pain phenotypes after nerve injury. In this study, we present a detailed morphological examination of mRNA expression in the DRG in the same mouse strains. For Nav 1.9, TRPA1, and TRPM8, the size spectra of labeled neurons remained mostly unchanged after injury in all strains. However, in CBA, AKR, and C58 mice, injury caused a preferential downregulation of Nav 1.8 in large diameter neurons. In CBA mice there was a shift toward larger neuronal profiles expressing TRPV1 after injury, indicating de novo (or upregulated) expression of TRPV1 in a subpopulation of neurons that normally does not express this gene. Finally, in C58 mice there was a shift toward smaller P2X3‐expressing neuronal profiles after injury, suggesting that a loss of P2X3 mRNA transcript occurred preferentially in medium‐sized cells. We used a multivariate statistical model to compare the regulation patterns of the six DRG genes. Clustering patterns suggested that genes of similar phylogenetic origin and function are regulated similarly.

Germfree status of mice obtained by embryo transfer in an isolator environment.

The technique of embryo transfer has been evaluated for the purpose of changing the mouse stocks to a germfree (GF) status. Our results show reproducible and quality-assured conversion of animals to those which are negative for the presence of microorganisms. Rapid and easy access to GF mice is advantageous for studies of selected microflora and their cross-talks with the host, when applying, e.g. genomic, proteomic and metabolic methodology. The study involved embryo transfer in an isolator environment, thereby allowing implantation of cleansed embryos into GF recipients under well-controlled conditions. The recipient females gave birth normally and took care of the offspring as if they were their own pups, thus enhancing the survival rate. Access to full technical resources required to maintain GF isolators are, however, a prerequisite. In this study, we used stainless steel isolators designed by Gustafsson (1959), on which a stereomicroscope was mounted to facilitate embryo transfer inside the isolator. The use of embryo transfer and isolator techniques will facilitate the availability of various mouse mutant models under different gnotobiotic conditions, GF, monoxenic or polyxenic animals, to enable comparison with conventional animals for physiological and pathophysiological studies.

Differential expression of neuronal voltage-gated sodium channel mRNAs during the development of the rat trigeminal ganglion.

The developmental pattern of sodium channel expression in neurons of primary sensory ganglia is likely reflected in the electrical behavior of these cells. Little information is available on how voltage-gated sodium channels in sensory neurons are expressed during development in the trigeminal-innervated craniofacial region, where sensitivity is fundamental during early stages of life. Using in situ hybridization, we here demonstrate a differential both regional and transcript-dependent distribution of sodium channel alpha- and beta-subunits between Embryonic day (E)15 and Postnatal day (P)5/6 in the rat trigeminal ganglion. Na(v)1.3 mRNA was strongly expressed at E15, but declined to low levels at P5/P6. Na(v)1.8 was expressed at E15, increased to reach maximum levels at P1 and then decreased. Na(v)1.9 mRNA was detected at E19, reached a maximum at P1, and was then reduced. beta1 mRNA showed a steady rise in expression from E17, while beta2 mRNA was widely expressed from P1. beta 3 mRNA was detected at E15, reached a maximum at E19 followed by a decrease in expression. In the ophthalmic TG portion, there was a higher expression level of Na(v)1.8 and Na(v)1.9 between E19 and P5/P6 as compared to the maxillary/mandibular part, indicating an unexpected positional difference in channel distribution. mRNA levels of p11, which facilitates the expression of Na(v)1.8, were high at all stages. These findings show that trigeminal ganglion sodium channel transcripts mature in steps that are specific for each transcript. They also raise the possibility that different facial regions could differ in the ability to transmit sensory signals during early life.

In vivo engineering of mobilized stem cell grafts with the immunomodulatory drug FTY720 for allogeneic transplantation

The immunological attributes of stem cell grafts play an important role in the outcome of allogeneic stem cell transplants. Currently, ex vivo manipulation techniques such as bulk T‐cell depletion or positive selection of CD34+ cells are utilized to improve the immunological attributes of grafts and minimize the potential for graft‐versus‐host disease (GvHD). Here, we demonstrate a novel graft engineering technique, which utilizes the immunomodulatory drug FTY720 for in vivo depletion of naïve T (TN) cells from donor G‐CSF‐mobilized grafts without ex vivo manipulation. We show that treatment of donor mice with FTY720 during mobilization depletes grafts of TN cells and prevents lethal GvHD following transplantation in a major mismatch setting. Importantly, both stem cells and NK cells are retained in the FTY720‐treated grafts. FTY720 treatment does not negatively affect the engraftment potential of stem cells as demonstrated in our congenic transplants or the functionality of NK cells. In addition, potentially useful memory T cells may be retained in the graft. These findings suggest that FTY720 may be used to optimize the immunological attributes of G‐CSF‐mobilized grafts by removing potentially deleterious TN cells which can contribute to GvHD, and by retaining useful cells which can promote immunity in the recipient.