Mapping lumbar efferent and afferent spinal circuitries via paddle array in a porcine model.

BACKGROUND: Preclinical models are essential for identifying changes occurring after neurologic injury and assessing therapeutic interventions. Yucatan miniature pigs (minipigs) have brain and spinal cord dimensions like humans and are useful for laboratory-to-clinic studies. Yet, little work has been done to map spinal sensorimotor distributions and identify similarities and differences between the porcine and human spinal cords. NEW METHODS: To characterize efferent and afferent signaling, we implanted a conventional 32-contact, four-column array into the dorsal epidural space over the lumbosacral spinal cord, spanning the L5-L6 vertebrae, in two Yucatan minipigs. Spinally evoked motor potentials were recorded bilaterally in four hindlimb muscles during stimulation delivered from different array locations. Then, cord dorsum potentials were recorded via the array by stimulating the left and right tibial nerves. RESULTS: Utilizing epidural spinal stimulation, we achieved selective left, right, proximal, and distal activation in the hindlimb muscles. We then determined the selectivity of each muscle as a function of stimulation location which relates to the distribution of the lumbar motor pools. COMPARISON WITH EXISTING METHODS: Mapping motoneuron distribution to hindlimb muscles and recording responses to peripheral nerve stimulation in the dorsal epidural space reveals insights into ascending and descending signal propagation in the lumbar spinal cord. Clinical-grade arrays have not been utilized in a porcine model. CONCLUSIONS: These results indicate that efferent and afferent spinal sensorimotor networks are spatially distinct, provide information about the organization of motor pools in the lumbar enlargement, and demonstrate the feasibility of using clinical-grade devices in large animal research.

Cortical stimulation leads to shortened myelin sheaths and increased axonal branching in spared axons after cervical spinal cord injury.

Neural activity and learning lead to myelin sheath plasticity in the intact central nervous system (CNS), but this plasticity has not been well-studied after CNS injury. In the context of spinal cord injury (SCI), demyelination occurs at the lesion site and natural remyelination of surviving axons can take months. To determine if neural activity modulates myelin and axon plasticity in the injured, adult CNS, we electrically stimulated the contralesional motor cortex at 10 Hz to drive neural activity in the corticospinal tract of rats with sub-chronic spinal contusion injuries. We quantified myelin and axonal characteristics by tracing corticospinal axons rostral to and at the lesion epicenter and identifying nodes of Ranvier by immunohistochemistry. Three weeks of daily stimulation induced very short myelin sheaths, axon branching, and thinner axons outside of the lesion zone, where remodeling has not previously been reported. Surprisingly, remodeling was particularly robust rostral to the injury which suggests that electrical stimulation can promote white matter plasticity even in areas not directly demyelinated by the contusion. Stimulation did not alter myelin or axons at the lesion site, which suggests that neuronal activity does not contribute to myelin remodeling near the injury in the sub-chronic period. These data are the first to demonstrate wide-scale remodeling of nodal and myelin structures of a mature, long-tract motor pathway in response to electrical stimulation. This finding suggests that neuromodulation promotes white matter plasticity in intact regions of pathways after injury and raises intriguing questions regarding the interplay between axonal and myelin plasticity.

A micro-LED implant and technique for optogenetic stimulation of the rat spinal cord.

To date, relatively few studies have used optogenetic stimulation to address basic science and therapeutic questions within the spinal cord. Even less have reported optogenetic stimulation in the rat spinal cord. This is likely due to a lack of accessible optogenetic implants. The development of a device that can be fabricated and operated by most laboratories, requiring no special equipment, would allow investigators to begin dissecting the functions of specific neuronal cell-types and circuitry within the spinal cord, as well as investigate therapies for spinal ailments like spinal cord injury. Here, we describe a long-term implantable µLED device designed for optogenetic stimulation of the spinal cord in awake, freely moving rats that is simple enough to be fabricated, implanted and operated by most laboratories. This device, which sits above the dorsal cord, can induce robust movements for at least 6 weeks without causing physical or thermal damage to the underlying spinal cord. In this regard, the presented µLED device could help tease apart the complexities of the spinal cord and uncover potential future therapeutics.

Optogenetic surface stimulation of the rat cervical spinal cord.

Electrical intraspinal microstimulation (ISMS) at various sites along the cervical spinal cord permits forelimb muscle activation, elicits complex limb movements and may enhance functional recovery after spinal cord injury. Here, we explore optogenetic spinal stimulation (OSS) as a less invasive and cell type-specific alternative to ISMS. To map forelimb muscle activation by OSS in rats, adeno-associated viruses (AAV) carrying the blue-light sensitive ion channels channelrhodopsin-2 (ChR2) and Chronos were injected into the cervical spinal cord at different depths and volumes. Following an AAV incubation period of several weeks, OSS-induced forelimb muscle activation and movements were assessed at 16 sites along the dorsal surface of the cervical spinal cord. Three distinct movement types were observed. We find that AAV injection volume and depth can be titrated to achieve OSS-based activation of several movements. Optical stimulation of the spinal cord is thus a promising method for dissecting the function of spinal circuitry and targeting therapies following injury. NEW & NOTEWORTHY Optogenetics in the spinal cord can be used both for therapeutic treatments and to uncover basic mechanisms of spinal cord physiology. For the first time, we describe the methodology and outcomes of optogenetic surface stimulation of the rat spinal cord. Specifically, we describe the evoked responses of forelimbs and address the effects of different adeno-associated virus injection paradigms. Additionally, we are the first to report on the limitations of light penetration through the rat spinal cord.

Proportion of Tubal Factor Infertility due to Chlamydia: Finite Mixture Modeling of Serum Antibody Titers.

In this study, we examined whether the proportion of tubal factor infertility (TFI) that is attributable to Chlamydia trachomatis, the population excess fraction (PEF), can be estimated from serological data using finite mixture modeling. Whole-cell inclusion immunofluorescence serum antibody titers were recorded among infertile women seen at St. Michael's Hospital in Bristol, United Kingdom, during the period 1985-1995. Women were classified as TFI cases or controls based on laparoscopic examination. Finite mixture models were used to identify the number of component titer distributions and the proportion of serum samples in each, from which estimates of PEF were derived. Four titer distributions were identified. The component at the highest titer was found only in samples from women with TFI, but there was also an excess of the second-highest titer component in TFI cases. Minimum and maximum estimates of the PEF were 28.0% (95% credible interval: 6.9, 50.0) and 46.8% (95% credible interval: 23.2, 64.1). Equivalent estimates based on the standard PEF formula from case-control studies were 0% and over 65%. Finite mixture modeling can be applied to serological data to obtain estimates of the proportion of reproductive damage attributable to C. trachomatis Further studies using modern assays in contemporary, representative populations should be undertaken.

Pelvic inflammatory disease and salpingitis: incidence of primary and repeat episodes in England.

Pelvic inflammatory disease (PID) and more specifically salpingitis (visually confirmed inflammation) is the primary cause of tubal factor infertility and is an important risk factor for ectopic pregnancy. The risk of these outcomes increases following repeated episodes of PID. We developed a homogenous discrete-time Markov model for the distribution of PID history in the UK. We used a Bayesian framework to fully propagate parameter uncertainty into the model outputs. We estimated the model parameters from routine data, prospective studies, and other sources. We estimated that for women aged 35-44 years, 33·6% and 16·1% have experienced at least one episode of PID and salpingitis, respectively (diagnosed or not) and 10·7% have experienced one salpingitis and no further PID episodes, 3·7% one salpingitis and one further PID episode, and 1·7% one salpingitis and ⩾2 further PID episodes. Results are consistent with numerous external data sources, but not all. Studies of the proportion of PID that is diagnosed, and the proportion of PIDs that are salpingitis together with the severity distribution in different diagnostic settings and of overlap between routine data sources of PID would be valuable.

MMP9-sensitive polymers mediate environmentally-responsive bivalirudin release and thrombin inhibition.

MMP9-responsive bivalirudin-HPMA copolymers were synthesized for direct, local administration in rat spinal cord contusion injury models. Polymer-conjugated bivalirudin peptides maintained activity while demonstrating enzyme-mediated release upon MMP9 exposure and prolonged release from hyaluronic acid/methylcellulose (HAMC) hydrogels compared to free bivalirudin peptide. Localized administration of bivalirudin copolymers in vivo at the site of rat spinal cord injury decreased cellular proliferation and astrogliosis, suggesting the bivalirudin copolymer and HAMC hydrogel system are a promising therapeutic intervention for reducing immediate inflammatory responses and long term scarring.

Incidence of Chlamydia trachomatis infection in women in England: two methods of estimation.

Information on the incidence of Chlamydia trachomatis (CT) is essential for models of the effectiveness and cost-effectiveness of screening programmes. We developed two independent estimates of CT incidence in women in England: one based on an incidence study, with estimates 'recalibrated' to the general population using data on setting-specific relative risks, and allowing for clearance and re-infection during follow-up; the second based on UK prevalence data, and information on the duration of CT infection. The consistency of independent sources of data on incidence, prevalence and duration, validates estimates of these parameters. Pooled estimates of the annual incidence rate in women aged 16-24 and 16-44 years for 2001-2005 using all these data were 0·05 [95% credible interval (CrI) 0·035-0·071] and 0·021 (95% CrI 0·015-0·028), respectively. Although, the estimates apply to England, similar methods could be used in other countries. The methods could be extended to dynamic models to synthesize, and assess the consistency of data on contact and transmission rates.

Presentation of clinically suspected persistent chlamydial infection: a case series.

In vivo antimicrobial resistance has yet to be documented in Chlamydia trachomatis; however, there have been anecdotal reports of persistent infection. The purpose of this case series was to describe a group of patients who have persistent chlamydia infection despite adequate treatment and where re-infection was considered unlikely. Patients were selected using a clinical questionnaire. For inclusion patients had to have tested positive for C. trachomatis, at least twice, using a nucleic acid amplification test despite having been fully compliant with at least two rounds of recommended therapy and be deemed to be at low risk of re-infection. Patients were grouped into categories based on sexual behaviour. Twenty-eight patients are included in this case series; 46% declared no sexual contact since initial diagnosis (category 1), a further 36% declaring contact that was considered low risk of re-infection (categories 2-4); 61% showed signs and symptoms at initial presentation increasing to 75% at re-attendance. Thirty-nine percent of patients received azithromycin only while 48% received doxycycline also. This case series identifies patients with persistent chlamydia despite receiving treatment. There is a need for a case definition of clinical treatment failure, development of susceptibility testing methods and guidance on appropriate treatment for patients with persistent infection.

Therapeutic intraspinal microstimulation improves forelimb function after cervical contusion injury.

OBJECTIVE: Intraspinal microstimulation (ISMS) is a promising method for activating the spinal cord distal to an injury. The objectives of this study were to examine the ability of chronically implanted stimulating wires within the cervical spinal cord to (1) directly produce forelimb movements, and (2) assess whether ISMS stimulation could improve subsequent volitional control of paretic extremities following injury. APPROACH: We developed a technique for implanting intraspinal stimulating electrodes within the cervical spinal cord segments C6-T1 of Long-Evans rats. Beginning 4 weeks after a severe cervical contusion injury at C4-C5, animals in the treatment condition received therapeutic ISMS 7 hours/day, 5 days/week for the following 12 weeks. MAIN RESULTS: Over 12 weeks of therapeutic ISMS, stimulus-evoked forelimb movements were relatively stable. We also explored whether therapeutic ISMS promoted recovery of forelimb reaching movements. Animals receiving daily therapeutic ISMS performed significantly better than unstimulated animals during behavioural tests conducted without stimulation. Quantitative video analysis of forelimb movements showed that stimulated animals performed better in the movements reinforced by stimulation, including extending the elbow to advance the forelimb and opening the digits. While threshold current to elicit forelimb movement gradually increased over time, no differences were observed between chronically stimulated and unstimulated electrodes suggesting that no additional tissue damage was produced by the electrical stimulation. SIGNIFICANCE: The results indicate that therapeutic intraspinal stimulation delivered via chronic microwire implants within the cervical spinal cord confers benefits extending beyond the period of stimulation, suggesting future strategies for neural devices to promote sustained recovery after injury.

Real-time quantitative PCR to determine chlamydial load in men and women in a community setting.

We used a PCR method to quantify the loads of Chlamydia trachomatis organisms in self-collected urine and vulvovaginal swab (VVS) samples from 93 women and 30 men participating in the Chlamydia Screening Studies Project, a community-based study of individuals not seeking health care. For women, self-collected VVS had a higher mean chlamydial load (10,405 organisms/ml; 95% confidence interval [95% CI], 5,167 to 21,163 organisms/ml) than did first-void urines (FVU) (503 organisms/ml; 95% CI, 250 to 1,022 organisms/ml; P < 0.001). Chlamydial loads in female and male self-collected FVU specimens were similar (P = 0.634). The mean chlamydial load in FVU specimens decreased with increasing age in females and males. There was no strong statistical evidence of differences in chlamydial load in repeat male and female FVU specimens taken when patients attended for treatment a median of 23.5 (range, 14 to 62) and 28 (range, 13 to 132) days later, respectively, or in VVS taken a median of 35 (range, 14 to 217) days later. In this study, chlamydial load values for infected persons in the community who were not seeking treatment were lower than those published in other studies involving symptomatic patients attending clinical settings. This might have implications for estimates of the infectiousness of chlamydia. The results of this study provide a scientific rationale for preferring VVS to FVU specimens from women.

Reactive nonproliferative gliosis predominates in a chronic mouse model of glaucoma.

In many CNS diseases, proliferation becomes dysregulated; cells divide and participate in pathological processes. Gliosis is a fundamental CNS response to trauma or disease in which cell hypertrophy and proliferation play prominent roles. The DBA/2J mouse is a glaucoma model in which mice experience gliosis concomitant with raised intraocular pressure that leads to a slow and progressive retinal ganglion cell axonopathy. We sought to determine if glaucomatous changes in DBA/2 retina would alter the regulation of cell proliferation, specifically in relation to retinal glia. Astrocyte and Müller glia populations within DBA/2 retina upregulated glial fibrillary acidic protein mRNA and protein compared with C57Bl/6; microglial cell number increased twofold from 4 to 10 months. Various bromodeoxyuridine (BrdU) injection paradigms were used to label dividing cells in DBA/2 and C57Bl/6 retina at 4 and 10 months of age. Very modest cell division in the retina, primarily in ganglion cell and inner nuclear layers, was observed at all ages. Immunohistochemistry indicated cell turnover primarily of NG2+ pericytes and Iba1+ microglia; astrocytes and Müller glia did not proliferate. There were no significant differences in BrdU+ cell numbers in 4 and 10-month-old retina, though 4-month retina had generally fewer BrdU+ cells than 10-month. C57Bl/6 retinas had fewer BrdU+ cells than DBA/2 retinas at all ages. These data show that, in contrast to gliosis in other CNS trauma and neurodegenerative diseases, glaucomatous changes in retina do not include substantive cell proliferation. Retinal changes in a chronic model of glaucoma engender a reactive, not proliferative, gliosis response.

Mycoplasma genitalium is associated with symptomatic urethritis.

In order to further investigate the epidemiology of Mycoplasma genitalium, 680 men attending departments of genitourinary medicine in Bristol, Bath and Truro were studied. M. genitalium was detected in 36 men (5.3%) and was present at all three clinics. Clinically, both urethritis and the presence of a urethral discharge and/or dysuria, but not penile irritation were independently associated with the detection of M. genitalium, the former being with the strongest association (odds ratio [OR] 10.76, 95% confidence interval [CI] [3.10-37.29], P < 0.0001; OR 3.01, 95% CI [1.28-7.05], P = 0.011 and OR 1.28, 95% CI [0.61-2.69], P = 0.51, respectively). In men with urethritis, those with a discharge and/or dysuria were more likely to have M. genitalium detected (OR 2.61, 95% CI [1.09-6.25], P = 0.032). We found no association with younger age or a recent change of sexual partner. In conclusion, M. genitalium is associated with symptomatic urethritis.

Should we still be testing for asymptomatic non-specific urethritis in departments of genitourinary medicine?

It has recently been advocated that non-invasive testing with first-catch urine specimens using nucleic acid amplification techniques, to detect Chlamydia trachomatis and Neisseria gonorrhoeae, should replace routine microscopy on asymptomatic men. Although it is assumed that this strategy will be cost effective, the available evidence suggests that this will result in fewer sexually transmitted infections being averted than continuing the current practice of screening for urethritis and testing for both microorganisms in asymptomatic men. This review article summarizes the available evidence and argues that research is urgently needed in order to properly evaluate the cost-effectiveness of detecting urethritis in asymptomatic men.

An oral fluid test for syphilis.

We have developed a time-resolved fluorescence immunoassay to detect antibodies to Treponema pallidum recombinant antigens in oral fluid specimens. Using an 'Oracol' swab, oral fluid was collected from 34 subjects with a serological diagnosis of syphilis and 97 seronegative controls. Using a cut-off of three standard deviations over control mean, the sensitivity and specificity of the assay in all subjects with positive syphilis serology was 76.5% and 97.9%, respectively. In those with early syphilis, the sensitivity and specificity of the assay was 100% and 97.9%. In a non-outbreak situation, screening clinic attendees for syphilis using oral fluid specimens is potentially useful when collection of blood is not practicable. In addition, it may have much to offer in outreach projects and epidemiological investigations.

The chlamydia screening studies: rationale and design.

BACKGROUND: Screening has been recommended to reduce the prevalence and morbidity associated with genital chlamydia infection in the United Kingdom. METHODS: We describe the rationale and study design of the Chlamydia Screening Studies (ClaSS), a collaborative project designed to evaluate screening outside genitourinary medicine clinics. A non-selective, active screening approach in 16-39 year olds randomly sampled from 27 general practice lists in the Bristol and Birmingham areas formed the basis of interlinked studies: a case-control study was used to investigate factors to improve the targeting of screening; participants with chlamydia were invited to enroll in a randomised controlled trial to evaluate partner notification conducted in primary care; and laboratory based studies were used to assess the best specimens and tests. We also explored psychosocial effects of screening and partner notification and modelled the cost effectiveness of the programme. CONCLUSION: Results from four pilot practices show that mailing of specimens for chlamydia testing is feasible but that it is difficult to achieve high response rates with postal screening. The high prevalence of asymptomatic infection in men suggests that efforts to screen men for chlamydia should be strengthened.

Diagnosis of Chlamydia trachomatis in North Thames region 2003.

A multi-centre re-audit of tests used for chlamydia diagnosis in GU medicine clinic attendees from February 2003 to March 2003 in the North Thames region showed improvements since our previous audit in 1999, with a significant increase in the proportion of clinics using nucleic acid amplification tests and non-invasive testing.

Treatment of Chlamydia trachomatis in North Thames region 2003.

Following on from the survey of techniques used for testing chlamydia, a multi-centre re-audit of the treatment of C. trachomatis in genitourinary clinic attendees in the North Thames region from February to March 2003 was performed. This showed an improvement since our previous audit with a significant increase in the number of centres following national guidelines in antibiotic prescribing and offering test of cure in clinically indicated cases.

A novel polymerase chain reaction assay to detect Mycoplasma genitalium.

AIMS: To design and validate a polymerase chain reaction (PCR) assay targeting the 16S rRNA gene of Mycoplasma genitalium. METHODS: Primers were designed that were complementary to the 16S rRNA gene sequence of M genitalium. After optimisation of the reaction conditions, the PCR was tested against nine M genitalium strains, a dilution series of M genitalium DNA, and a panel of common microorganisms. The PCR was also challenged in parallel with a published assay against 54 urine specimens from men with urethritis. RESULTS: The expected 341 bp product was produced on amplification of material from all M genitalium strains and from none of the other microorganisms tested. The lower limit of detection was 50 genome copies. The new assay detected M genitalium DNA in nine of 54 men with urethritis, in comparison with eight positive specimens detected with the alternative PCR. CONCLUSIONS: This novel PCR targeting the M genitalium 16S rRNA gene has been optimised and now provides a sensitive and specific alternative or addition to the available MgPa gene targeting assays.