Startling Acoustic Stimulation Has Task-Specific Effects on Intracortical Facilitation and Inhibition at Rest and During Visually Guided Isometric Elbow Flexion in Healthy Individuals.

Startling acoustic stimulation (SAS) causes a transient effect on the primary motor cortex (M1) nonreflexively. It reduces the cortical excitability at rest, but not during voluntary contraction. However, the effect of SAS on intracortical activity is not clear. The purpose of this study was to investigate the SAS effect on short-interval intracortical inhibition and intracortical facilitation using transcranial magnetic stimulation (TMS). Eleven healthy individuals performed isometric elbow flexion at 10% of maximum voluntary contraction on the dominant side with a real-time visual target (i.e., M1 preactivation) or at rest. TMS was delivered to the M1 ipsilateral to elbow flexion without or with SAS delivered 90 ms prior to TMS. There were three TMS delivery conditions: (a) single pulse, (b) short-interval intracortical inhibition, and (c) intracortical facilitation. TMS-induced motor-evoked potential (MEP) was compared between predetermined TMS and SAS conditions at rest and during ipsilateral voluntary contraction. We confirmed that SAS decreased the MEP amplitude at rest, but not during M1 preactivation. SAS caused task-specific effects on intracortical excitability. Specifically, SAS increased intracortical facilitation at rest and during voluntary contraction. However, SAS decreased short-interval intracortical inhibition only during M1 preactivation. Collectively, our results suggest that SAS transiently influences the motor cortex excitability, possibly via its activation of higher centers, to achieve a visually guided goal-directed task.

Observations of Autonomic Variability Following Central Neuromodulation for Chronic Neuropathic Pain in Spinal Cord Injury.

BACKGROUND: Spinal cord injury (SCI) persons with chronic neuropathic pain (NP) demonstrate maladaptive autonomic profiles compared to SCI counterparts without NP (SCI - NP) or able-bodied (AB) controls. These aberrations may be secondary to maladaptive neuroplasticity in the shared circuitry of the pain neuromatrix-central autonomic network interface (PNM-CAN). In this study, we explored the proposed PNM-CAN mechanism in SCI + NP and AB cohorts following centrally-directed neuromodulation to assess if the PNM and CAN are capable of being differentially modulated. MATERIALS AND METHODS: Central neuromodulation was administered via breathing-controlled electrical stimulation (BreEStim), previously evidenced to operate at the PNM. To quantify CAN activity, conventional heart rate variability (HRV) recordings were used to gather time and frequency domain parameters of autonomic modulation. SCI + NP (n = 10) and AB (n = 13) cohorts received null and active BreEStim randomly in crossover fashion. HRV data were gathered pretest and 30 minutes posttest. Pain modulation was quantified at both time-points by visual analog scale (VAS) for SCI + NP persons and electrical detection and pain threshold levels (EDT, EPT) for AB persons. RESULTS: Following active BreEStim only, SCI + NP persons demonstrated increased parasympathetic tone (increased NN50, p = 0.03, and pNN50, p = 0.02, HRV parameters). This parasympathetic restoration was associated with analgesia (VAS reduction, p < 0.01). Similarly, AB persons demonstrated increased noxious tolerance (increased EPT, p = 0.03, with preserved EDL, p = 0.78) only following active BreEStim. However, this increased pain threshold was not associated with autonomic changes. CONCLUSIONS: Central modulation targeting the PNM produced autonomic changes in SCI + NP persons but not AB persons. These findings suggest that AB persons exhibit intact CAN mechanisms capable of compensating for PNM aberrations or simply that SCI + NP persons exhibit altered PNM-CAN machinery altogether. Our collective findings confirm the interconnectedness and maladaptive plasticity of PNM-CAN machinery in SCI + NP persons and suggest that the PNM and CAN circuitry can be differentially modulated.

The effects of conditioning startling acoustic stimulation (SAS) on the corticospinal motor system: a SAS-TMS study.

A startling acoustic stimulus (SAS) could cause transient effects on the primary motor cortex and its descending tracts after habituation of reflex responses. In the literature, there is evidence that the effects of SAS depend on the status of M1 excitability and delivery time of SAS. In this study, we aimed to comprehensively investigate the effects of SAS on the excitability of primary motor cortex. Eleven healthy subjects participated in this study. Transcranial magnetic stimulation (TMS) was delivered to the hot spot for left biceps at rest and during isometric right elbow flexion (10, 30, and 60% of their maximum voluntary contraction, MVC). There were three SAS conditions: (1) No SAS; (2) SAS was delivered 50 ms prior to TMS (SAS50); (3) SAS 90 ms prior to TMS (SAS90). For each subject, the induced MEP amplitude was normalized to the largest response at rest with No SAS. Two-way ANOVAs (4 force levels × 3 SAS conditions) with repeated measures were used to determine the differences under different conditions. For the MEP amplitude, there were significant force level effect and FORCE LEVEL × SAS interactions. Specifically, the MEP amplitude increased with force level. Furthermore, post hoc analysis showed that the MEP amplitude reduced during SAS50 and SAS90 compared to No SAS only at rest. Our results provide evidence that a conditioning SAS causes a transient suppression of the corticospinal excitability at rest when it is delivered 50 ms and 90 ms prior to TMS. However, a conditioning SAS has no effect when the corticospinal excitability is already elevated with an external visual target.

A startling acoustic stimulation (SAS)-TMS approach to assess the reticulospinal system in healthy and stroke subjects.

Reticulospinal (RS) hyperexcitability is observed in stroke survivors with spastic hemiparesis. Habituated startle acoustic stimuli (SAS) can be used to stimulate the RS pathways non-reflexively. However, the role of RS pathways in motor function and its interactions with the corticospinal system after stroke still remain unclear. Therefore, the purpose of this study was to investigate the effects of conditioning SAS on the corticospinal system in healthy subjects and in stroke subjects with spastic hemiparesis. An established conditioning SAS- transcranial magnetic stimulation (TMS) paradigm was used to test the interactions between the RS pathways and the corticospinal system. TMS was delivered to the right hemisphere of eleven healthy subjects and the contralesional hemisphere of eleven stroke subjects during isometric elbow flexor contraction on the non-impaired (or left) side. Conditioning SAS had similar effects on the corticospinal motor system in both healthy and stroke subjects, including similar SAS-induced motor evoked potential (MEP) reduction at rest, but not during voluntary contraction tasks; similar magnitudes of TMS-induced MEP and force increment and shortening of the silent period during voluntary elbow flexor contraction. This study provides evidence that RS excitability on the contralesional side in stroke subjects with spastic hemiparesis is not abnormal, and suggests that RS projections are likely to be primarily unilateral in humans.

Possible Contributions of Ipsilateral Pathways From the Contralesional Motor Cortex to the Voluntary Contraction of the Spastic Elbow Flexors in Stroke Survivors: A TMS Study.

OBJECTIVE: The contribution of the contralesional motor cortex to the impaired limbs is still controversial. The aim of this study was to investigate the role of descending projections from the contralesional hemisphere during voluntary elbow flexion on the paretic side. DESIGN: Eleven healthy and 10 stroke subjects performed unilateral isometric elbow flexion tasks at various submaximal levels. Transcranial magnetic stimulation was delivered to the hotspot of biceps muscles ipsilateral to the target side (paretic side in stroke subjects or right side in controls) at rest and during elbow flexion tasks. Motor-evoked potential amplitudes of the contralateral resting biceps muscles, transcranial magnetic stimulation-induced ipsilateral force increment, and reflex torque and weakness of spastic elbow flexors were quantified. RESULTS: The normalized motor-evoked potential amplitude increased with force level in both healthy and stroke subjects. However, stroke subjects exhibited significantly higher force increment compared with healthy subjects only at low level of elbow flexion but similar at moderate to high levels. The greater force increment significantly correlated with reflex torque of the spastic elbow flexors, but not weakness. CONCLUSIONS: These results provide novel evidence that ipsilateral projections are not likely to contribute to strength but are correlated to spasticity of spastic-paretic elbow flexors after stroke.

Motor Overflow and Spasticity in Chronic Stroke Share a Common Pathophysiological Process: Analysis of Within-Limb and Between-Limb EMG-EMG Coherence.

The phenomenon of exaggerated motor overflow is well documented in stroke survivors with spasticity. However, the mechanism underlying the abnormal motor overflow remains unclear. In this study, we aimed to investigate the possible mechanisms behind abnormal motor overflow and its possible relations with post-stroke spasticity. 11 stroke patients (63.6 ± 6.4 yrs; 4 women) and 11 healthy subjects (31.18 ± 6.18 yrs; 2 women) were recruited. All of them were asked to perform unilateral isometric elbow flexion at submaximal levels (10, 30, and 60% of maximum voluntary contraction). Electromyogram (EMG) was measured from the contracting biceps (iBiceps) muscle and resting contralateral biceps (cBiceps), ipsilateral flexor digitorum superficialis (iFDS), and contralateral FDS (cFDS) muscles. Motor overflow was quantified as the normalized EMG of the resting muscles. The severity of motor impairment was quantified through reflex torque (spasticity) and weakness. EMG-EMG coherence was calculated between the contracting muscle and each of the resting muscles. During elbow flexion on the impaired side, stroke subjects exhibited significant higher motor overflow to the iFDS muscle compared with healthy subjects (ipsilateral or intralimb motor overflow). Stroke subjects exhibited significantly higher motor overflow to the contralateral spastic muscles (cBiceps and cFDS) during elbow flexion on the non-impaired side (contralateral or interlimb motor overflow), compared with healthy subjects. Moreover, there was significantly high EMG-EMG coherence in the alpha band (6-12 Hz) between the contracting muscle and all other resting muscles during elbow flexion on the non-impaired side. Our results of diffuse ipsilateral and contralateral motor overflow with EMG-EMG coherence in the alpha band suggest subcortical origins of motor overflow. Furthermore, correlation between contralateral motor overflow to contralateral spastic elbow and finger flexors and their spasticity was consistently at moderate to high levels. A high correlation suggests that diffuse motor overflow to the impaired side and spasticity likely share a common pathophysiological process. Possible mechanisms are discussed.

Neuropathic pain modulation after spinal cord injury by breathing-controlled electrical stimulation (BreEStim) is associated with restoration of autonomic dysfunction.

BACKGROUND: Recent findings have implicated supraspinal origins from the pain neuromatrix- central autonomic network (PNM-CAN) in the generation of neuropathic pain (NP) after spinal cord injury (SCI). The aim of this study was to further investigate the theorized PNM-CAN mechanisms in persons with SCI by using a centrally directed pain intervention, provided by breathing-controlled electrical stimulation (BreEStim), to measure resultant autonomic changes measured by time and frequency domain heart rate variability (HRV) analysis. METHODS: Null and active BreEStim interventions were administered to SCI+NP subjects (n=10) in a random order. HRV data and VAS pain scores were collected at resting pre-test and 30 minutes post-test time points. Resting HRV data were also collected from SCI-NP subjects (n=11). RESULTS: SCI+NP subjects demonstrated a lower baseline HRV and parasympathetic tone, via SD of the normal-to-normal intervals (SDNN) and low frequency (LF) parameters, compared with SCI-NP subjects. However, following active BreEStim, SCI+NP subjects exhibited an increase in HRV and parasympathetic tone, most notably via pairs of successive R-R beat lengths varying by greater than 50 ms (NN50) and proportion of NN50 for total number of beats (pNN50) parameters along with lower VAS scores. Additionally, the post-test SCI+NP group was found to have a statistically comparable autonomic profile to the SCI-NP group across all HRV variables, including SDNN and LF parameters. CONCLUSION: The analgesic effects of active BreEStim in SCI+NP subjects were associated with restoration of autonomic dysfunction in this population.

Combined transcranial direct current stimulation and breathing-controlled electrical stimulation for management of neuropathic pain after spinal cord injury.

OBJECTIVE: To determine whether transcranial direct current stimulation augments the analgesic effect of breathing-controlled electrical stimulation in patients with spinal cord injury who have chronic neuropathic pain. DESIGN: Sham-controlled, single-blinded, single-centre, cross-over study of 12 participants with incomplete spinal cord injury. The treatment protocol included a 20-min transcranial direct current stimulation (sham or active), followed by a 20-min breathing-controlled electrical stimulation to the median nerve on the dominant side. The treatment session with sham or control transcranial direct current stimulation was given on different days in a randomized order. Visual analogue scale was used to assess neuropathic pain at baseline, 10 min after transcranial direct current stimulation, and 10 min after breathing-controlled electrical stimulation. RESULTS: Participants were blinded to the status of transcranial direct current stimulation. Out of the 12 participants, 10 completed sessions of both sham and active transcranial direct current stimulation, while the other 2 completed only active transcranial direct current stimulation and breathing-controlled electrical stimulation treatment. Out of the 12 participants, 7 showed analgesic effects after active transcranial direct current stimulation, while sham transcranial direct current stimulation produced some analgesic effects in 4 out of 10 participants. At the group level, there was no difference between active and sham transcranial direct current stimulation treatment. All except one participant responded positively to breathing-controlled electrical stimulation in all sessions. Visual analogue scale score for pain decreased significantly after breathing-controlled electrical stimulation combined with either active transcranial direct current stimulation or sham transcranial direct current stimulation treatment. CONCLUSION: The immediate analgesic effect of breathing-controlled electrical stimulation was confirmed. However, this effect was not augmented after one session of transcranial direct current stimulation treatment.

Heart Rate Variability: A Novel Modality for Diagnosing Neuropathic Pain after Spinal Cord Injury.

Background: Heart rate variability (HRV), the physiological variance in the heart's R-R interval length, can be analyzed to produce various parameters reflective of one's autonomic balance. HRV analysis may be used to capture those autonomic aberrations associated with chronic neuropathic pain (NP) in spinal cord injury (SCI). This study assesses the capacity of HRV parameters to diagnose NP in an SCI cohort. Methods: An electrocardiogram (ECG) was collected at rest from able bodied participants (AB, n = 15), participants with SCI only (SCI-NP, n = 11), and those with SCI and NP (SCI+NP, n = 20). HRV parameters were analyzed using conventional time and frequency analysis. Results: At rest, there were no heart rate differences amongst groups. However, SCI+NP participants demonstrated lower overall HRV, as determined by the SDNN time domain parameter, compared to either AB (p < 0.01) or SCI-NP (p < 0.05) groups. Moreover, AB and SCI-NP participants were statistically comparable for all HRV time and frequency domain parameters. Additional analyses demonstrated no differences in HRV parameters between T4, above vs. T5, below SCI groups (for all parameters: p > 0.15) or between C8, above vs. T1, below SCI groups (p > 0.30). Conclusions: Participants with SCI and NP exhibit a lower overall HRV, which can be determined by HRV time domain parameter SDNN. HRV analysis is an innovative modality with the capacity for objective quantification of chronic NP in participants with SCI.

A novel nonpharmacological intervention - breathing-controlled electrical stimulation for neuropathic pain management after spinal cord injury - a preliminary study.

OBJECTIVE: The objective of this study was to examine the effectiveness of a novel nonpharmacological intervention - breathing-controlled electrical stimulation (BreEStim) - for neuropathic pain management in spinal cord injury (SCI) patients. SUBJECTS AND METHODS: There were two experiments: 1) to compare the effectiveness between BreEStim and conventional electrical stimulation (EStim) in Experiment (Exp) 1 and 2) to examine the dose-response effect of BreEStim in Exp 2. In Exp 1, 13 SCI subjects (6 males and 7 females, history of SCI: 58.2 months, from 7 to 150 months, impairments ranging from C4 AIS B to L1 AIS B) received both BreEStim and EStim in a randomized order with at least 3 days apart. A total of 120 electrical stimuli to the median nerve transcutaneously were triggered by voluntary inhalation during BreEStim or were randomly delivered during EStim. In Exp 2, a subset of 7 subjects received BreEStim120 and 240 stimuli randomly on two different days with 7 days apart (BreEStim120 vs BreEStim240). The primary outcome variable was the visual analog scale (VAS) score. RESULTS: In Exp 1, both BreEStim and EStim showed significant analgesic effects. Reduction in VAS score was significantly greater after BreEStim (2.6±0.3) than after EStim (0.8±0.3) (P<0.001). The duration of analgesic effect was significantly longer after BreEStim (14.2±6 hours) than after EStim (1.9±1 hours) (P=0.04). In Exp 2, BreEStim120 and BreEStim240 had similar degree and duration of analgesic effects. CONCLUSION: The findings from this preliminary study suggest that BreEStim is an effective alternative nonpharmacological treatment for chronic neuropathic pain in patients suffering from SCI.

Different Effects of Startling Acoustic Stimuli (SAS) on TMS-Induced Responses at Rest and during Sustained Voluntary Contraction.

Previous studies have shown that a habituated startling acoustic stimulus (SAS) can cause a transient suppression of motor evoked potentials (MEPs) induced by transcranial magnetic stimulation (TMS) during light muscle contraction. However, it is still unknown whether this phenomenon persists when at rest or during a sustained voluntary contraction task. Therefore, the purpose of this study was to determine whether a conditioning SAS has different effects. TMS was delivered to the hot spot for the left biceps on 11 subjects at rest both with and without a conditioning SAS. Of the 11subjects, 9 also had TMS delivered during isometric flexion of the left elbow, also with and without a conditioning SAS. TMS-induced MEPs, TMS-induced force, and silent periods were used to determine the effect of conditioning SAS. Consistent with previous findings, TMS-induced MEPs were smaller with a conditioning SAS (0.49 ± 0.37 mV) as compared without the SAS (0.69 ± 0.52 mV) at rest. However, a conditioning SAS during the voluntary contraction tasks resulted in a significant shortening of the MEP silent period (187.22 ± 22.99 ms with SAS vs. 200.56 ± 29.71 ms without SAS) without any changes in the amplitude of the MEP (1.37 ± 0.9 mV with SAS V.S. 1.32 ± 0.92 mV without SAS) or the TMS-induced force (3.11 ± 2.03 N-m with SAS V.S. 3.62 ± 1.33 N-m without SAS). Our results provide novel evidence that a conditioning SAS has different effects on the excitability of the motor cortex when at rest or during sustained voluntary contractions.

Pain modulation effect of breathing-controlled electrical stimulation (BreEStim) is not likely to be mediated by deep and fast voluntary breathing.

Voluntary breathing-controlled electrical stimulation (BreEStim), a novel non-invasive and non-pharmacological treatment protocol for neuropathic pain management, was reported to selectively reduce the affective component of pain possibly by increasing pain threshold. The underlying mechanisms involved in the analgesic effect of BreEStim were considered to result from combination of multiple internal pain coping mechanisms triggered during BreEStim. Findings from our recent studies have excluded possible roles of acupuncture and aversiveness and habituation of painful electrical stimulation in mediating the analgesia effect of BreEStim. To further investigate the possible role of voluntary breathing during BreEStim, the effectiveness of fast and deep voluntary breathing-only and BreEStim on experimentally induced pain was compared in healthy human subjects. Results showed no change in electrical pain threshold after Breathing-only, but a significant increase in electrical pain threshold after BreEStim. There was no statistically significant change in other thresholds after Breathing-only and BreEStim. The findings suggest that the analgesic effect of BreEStim is not likely attributed to fast and deep voluntary breathing. Possible mechanisms are discussed.

Tactile, thermal, and electrical thresholds in patients with and without phantom limb pain after traumatic lower limb amputation.

PURPOSE: To examine whether there is central sensitization in patients with phantom limb pain (PLP) after traumatic limb amputation. METHODS: Seventeen patients after unilateral lower limb amputation secondary to trauma were enrolled. Ten patients had chronic PLP, while the other seven patients had no PLP. Tactile-sensation threshold, cold- and warm-sensation thresholds, cold- and heat-pain thresholds, electrical-sensation threshold (EST), and electrical-pain threshold on the distal residual limb and the symmetrical site on the sound limb were measured in all tested patients. Their thresholds were compared within the PLP and non-PLP group, and between the groups. RESULTS: The novel findings included: 1) electrical-pain threshold was only decreased in the sound limb in the PLP group and there was no difference between two limbs in the non-PLP group, suggesting central sensitization in patients with PLP; and 2) EST was increased on the affected limb as compared to the sound limb within the PLP group, but there were no significant differences in EST between the PLP and non-PLP group. There were in general no significant differences in other tested thresholds within the groups and between groups. CONCLUSION: Our results demonstrate central sensitization in the patients with PLP after traumatic limb amputation.

Habituation to experimentally induced electrical pain during voluntary-breathing controlled electrical stimulation (BreEStim).

OBJECTIVE: Painful peripheral electrical stimulation to acupuncture points was found to cause sensitization if delivered randomly (EStim), but induced habituation if triggered by voluntary breathing (BreEStim). The objective was to systematically compare the effectiveness of BreEStim and EStim and to investigate the possible mechanisms mediating the habituation effect of BreEStim. METHODS: Eleven pain-free, healthy subjects (6 males, 5 females) participated in the study. Each subject received the BreEStim and EStim treatments in a random order at least three days apart. Both treatments consisted of 120 painful but tolerable stimuli to the ulnar nerve at the elbow on the dominant arm. BreEStim was triggered by voluntary breathing while EStim was delivered randomly. Electrical sensation threshold (EST) and electrical pain threshold (EPT) were measured from the thenar and hypothenar eminences on both hands at pre-intervention and 10-minutes post-intervention. RESULTS: There was no difference in the pre-intervention baseline measurement of EST and EPT between BreEStim and EStim. BreEStim increased EPT in all tested sites on both hands, while EStim increased EPT in the dominant hypothenar eminence distal to the stimulating site and had no effect on EPT in other sites. There was no difference in the intensity of electrical stimulation between EStim and BreEStim. CONCLUSION: Our findings support the important role human voluntary breathing plays in the systemic habituation effect of BreEStim to peripheral painful electrical stimulation.

Modification of electrical pain threshold by voluntary breathing-controlled electrical stimulation (BreEStim) in healthy subjects.

BACKGROUND: Pain has a distinct sensory and affective (i.e., unpleasantness) component. BreEStim, during which electrical stimulation is delivered during voluntary breathing, has been shown to selectively reduce the affective component of post-amputation phantom pain. The objective was to examine whether BreEStim increases pain threshold such that subjects could have improved tolerance of sensation of painful stimuli. METHODS: Eleven pain-free healthy subjects (7 males, 4 females) participated in the study. All subjects received BreEStim (100 stimuli) and conventional electrical stimulation (EStim, 100 stimuli) to two acupuncture points (Neiguan and Weiguan) of the dominant hand in a random order. The two different treatments were provided at least three days apart. Painful, but tolerable electrical stimuli were delivered randomly during EStim, but were triggered by effortful inhalation during BreEStim. Measurements of tactile sensation threshold, electrical sensation and electrical pain thresholds, thermal (cold sensation, warm sensation, cold pain and heat pain) thresholds were recorded from the thenar eminence of both hands. These measurements were taken pre-intervention and 10-min post-intervention. RESULTS: There was no difference in the pre-intervention baseline measurement of all thresholds between BreEStim and EStim. The electrical pain threshold significantly increased after BreEStim (27.5±6.7% for the dominant hand and 28.5±10.8% for the non-dominant hand, respectively). The electrical pain threshold significantly decreased after EStim (9.1±2.8% for the dominant hand and 10.2±4.6% for the non-dominant hand, respectively) (F[1, 10] = 30.992, p = .00024). There was no statistically significant change in other thresholds after BreEStim and EStim. The intensity of electrical stimuli was progressively increased, but no difference was found between BreEStim and EStim. CONCLUSION: Voluntary breathing controlled electrical stimulation selectively increases electrical pain threshold, while conventional electrical stimulation selectively decreases electrical pain threshold. This may translate into improved pain control.

Alteration of fibroblast phenotype by asbestos-induced autoantibodies.

Pulmonary fibrosis is a relentlessly progressive disease for which the etiology can be idiopathic or associated with environmental or occupational exposures. There is not a clear explanation for the chronic and progressive nature of the disease, leaving treatment and prevention options limited. However, there is increasing evidence of an autoimmune component, since fibrotic diseases are often accompanied by production of autoantibodies. Because exposure to silicates such as silica and asbestos can lead to both autoantibodies and pulmonary/pleural fibrosis, these exposures provide an excellent tool for examining the relationship between these outcomes. This study explored the possibility that autoantibodies induced by asbestos exposure in mice would affect fibroblast phenotype. L929 fibroblasts and primary lung fibroblasts were treated with serum IgG from asbestos- or saline-treated mice, and tested for binding using cell-based ELISA, and for phenotypic changes using immunofluorescence, laser scanning cytometry and Sirius Red collagen assay. Autoantibodies in the serum of C57Bl/6 mice exposed to asbestos (but not sera from untreated mice) bound to mouse fibroblasts. The autoantibodies induced differentiation to a myofibroblast phenotype, as demonstrated by increased expression of smooth muscle α-actin (SMA), which was lost when the serum was cleared of IgG. Cells treated with purified IgG of exposed mice produced excess collagen. Using ELISA, we tested serum antibody binding to DNA topoisomerase (Topo) I, vimentin, TGFß-R, and PDGF-Rα. Antibodies to DNA Topo I and to PDGF-Rα were detected, both of which have been shown by others to be able to affect fibroblast phenotype. The anti-fibroblast antibodies (AFA) also induced STAT-1 activation, implicating the PDGF-R pathway as part of the response to AFA binding. These data support the hypothesis that asbestos induces AFA that modify fibroblast phenotype, and suggest a mechanism whereby autoantibodies may mediate some of the fibrotic manifestations of asbestos exposure.

Asbestos-induced autoimmunity in C57BL/6 mice.

Environmental impacts on autoimmunity have significant public health implications. Epidemiological studies have shown associations between exposure to airborne silicates, such as crystalline silica or asbestos, and autoimmunity, but the etiology remains unclear. The purpose of this study was to test the hypothesis that asbestos could lead to a specific pattern of autoantibodies and pathology indicative of systemic autoimmune disease (SAID). Female C57Bl/6 mice were instilled intratracheally with 2 doses x 60 microg/mouse of amphibole asbestos (tremolite), wollastonite (a non-fibrogenic control fiber), or saline alone. Serum samples were collected and urine was checked for protein bi-weekly for 7 months. By 26 weeks, the asbestos-instilled animals had a significantly higher frequency of positive anti-nuclear antibody (ANA) tests compared to wollastonite and saline groups. The majority of positive ANAs showed homogeneous or combined homogeneous/speckled patterns, and tested positive for antibodies to dsDNA and SSA/Ro 52. Serum isotyping showed no significant changes in IgM, IgA, or IgG subclasses. However, there was an overall decrease in the mean IgG serum concentration in asbestos-instilled mice. IgG immune complex deposition was demonstrated in the kidneys of asbestos-instilled mice, with evidence of glomerular and tubule abnormalities suggestive of glomerulonephritis. Flow cytometry demonstrated moderate changes in the percentages of CD25+ T-suppressor cells and B1a B-cells in the superficial cervical lymph nodes of the asbestos-instilled mice. These data demonstrate that asbestos leads to immunologic changes consistent with the development of autoimmunity. This study provides a non-autoimmune prone murine model for use in future elucidation of mechanisms involved in asbestos-induced autoimmune disease.

PERK eIF2alpha kinase regulates neonatal growth by controlling the expression of circulating insulin-like growth factor-I derived from the liver.

Humans afflicted with the Wolcott-Rallison syndrome and mice deficient for PERK (pancreatic endoplasmic reticulum eIF2alpha kinase) show severe postnatal growth retardation. In mice, growth retardation in Perk-/- mutants is manifested within the first few days of neonatal development. Growth parameters of Perk-/- mice, including comparison of body weight to length and organ weights, are consistent with proportional dwarfism. Tibia growth plates exhibited a reduction in proliferative and hypertrophic chondrocytes underlying the longitudinal growth retardation. Neonatal Perk-/- deficient mice show a 75% reduction in liver IGF-I mRNA and serum IGF-I within the first week, whereas the expression of IGF-I mRNA in most other tissues is normal. Injections of IGF-I partially reversed the growth retardation of the Perk-/- mice, whereas GH had no effect. Transgenic rescue of PERK activity in the insulin- secreting beta-cells of the Perk-/- mice reversed the juvenile but not the neonatal growth retardation. We provide evidence that circulating IGF-I is derived from neonatal liver but is independent of GH at this stage. We propose that PERK is required to regulate the expression of IGF-I in the liver during the neonatal period, when IGF-I expression is GH-independent, and that the lack of this regulation results in severe neonatal growth retardation.

The PERK eukaryotic initiation factor 2 alpha kinase is required for the development of the skeletal system, postnatal growth, and the function and viability of the pancreas.

Phosphorylation of eukaryotic initiation factor 2 alpha (eIF-2 alpha) is typically associated with stress responses and causes a reduction in protein synthesis. However, we found high phosphorylated eIF-2 alpha (eIF-2 alpha[P]) levels in nonstressed pancreata of mice. Administration of glucose stimulated a rapid dephosphorylation of eIF-2 alpha. Among the four eIF-2 alpha kinases present in mammals, PERK is most highly expressed in the pancreas, suggesting that it may be responsible for the high eIF-2 alpha[P] levels found therein. We describe a Perk knockout mutation in mice. Pancreata of Perk(-/-) mice are morphologically and functionally normal at birth, but the islets of Langerhans progressively degenerate, resulting in loss of insulin-secreting beta cells and development of diabetes mellitus, followed later by loss of glucagon-secreting alpha cells. The exocrine pancreas exhibits a reduction in the synthesis of several major digestive enzymes and succumbs to massive apoptosis after the fourth postnatal week. Perk(-/-) mice also exhibit skeletal dysplasias at birth and postnatal growth retardation. Skeletal defects include deficient mineralization, osteoporosis, and abnormal compact bone development. The skeletal and pancreatic defects are associated with defects in the rough endoplasmic reticulum of the major secretory cells that comprise the skeletal system and pancreas. The skeletal, pancreatic, and growth defects are similar to those seen in human Wolcott-Rallison syndrome.