Lumbosacral Cerebrospinal Fluid Research Articles

Comparison of atlantoaxial and lumbosacral cerebrospinal fluid centesis techniques in South American camelids.

Iatrogenic blood contamination during cerebrospinal fluid (CSF) centesis is common, which can limit the diagnostic usefulness of the sample. A novel ultrasound-guided CSF collection technique is described in horses, by which CSF is obtained from the atlantoaxial (AA) space. To compare ultrasound-guided AA centesis with lumbosacral (LS) centesis in South American camelids (SAC). The hypotheses were that AA centesis would yield samples with less blood contamination although being technically more challenging than LS centesis. Eight clinically healthy adult SAC from a university-owned teaching herd. Single-blinded, randomized, 4-way, 4-period crossover study in which 2 veterinarians each performed both centesis techniques on each animal once. Cytological sample analysis was performed, and the technical difficulty of sample acquisition was assessed. The CSF was collected successfully and without complications by either technique during all collection attempts. Aspects of technical difficulty and concentrations of CSF analytes did not vary significantly between techniques. Median total nucleated cell and red blood cell counts were 1/μL and 0.5/μL and 167.5/μL and 155/μL for AA and LS techniques, respectively. The median total protein concentration was 32.9 mg/dL and 38 mg/dL for AA and LS centeses. A median of 1 attempt was necessary for both centesis techniques and the median number of needle repositioning events was 1 for AA and 0 for LS. Depending on clinical circumstances, ultrasound-guided AA centesis appears to be an acceptable alternative to other techniques for collection of CSF from SAC.

Influence of laminectomy on the lumbosacral cerebrospinal fluid volume: A retrospective magnetic resonance imaging study.

The cerebrospinal fluid volume affects the block height of spinal anaesthesia. Laminectomy of the lumbar spine may result in increased lumbosacral cerebrospinal fluid volume. This study aimed to test the hypothesis that the lumbosacral cerebrospinal fluid volume of patients with a history of lumbar laminectomy would be larger than that of patients with normal lumbar spine anatomy using magnetic resonance imaging. Lumbosacral spine magnetic resonance images of 147 patients who underwent laminectomy at the L2 vertebrae or below (laminectomy group) and 115 patients without a history of spinal surgery (control group) were retrospectively reviewed. The lumbosacral cerebrospinal fluid volumes between the L1-L2 intervertebral disc level and the end of the dural sac were measured and compared between the two groups. The mean (standard deviation) lumbosacral cerebrospinal fluid volume was 22.3 (7.8) ml and 21.1 (7.4) ml in the laminectomy and control groups, respectively (mean difference 1.2 ml; 95% confidence interval -0.7 to 3.0 ml; P = 0.218). In the prespecified subgroup analysis according to the number of laminectomy levels, patients who underwent more than two levels of laminectomy exhibited slightly larger lumbosacral cerebrospinal fluid volume (n = 17, 30.5 (13.5) ml) compared with those who underwent two (n = 40, 20.7 (5.6) ml; P = 0.014) or one level of laminectomy (n = 90, 21.4 (6.2) ml; P = 0.010) and the control group (21.1 (7.4) ml; P = 0.012). In conclusion, the lumbosacral cerebrospinal fluid volume did not differ between patients who underwent lumbar laminectomy and those without a history of laminectomy. However, patients who underwent laminectomy at more than two levels had a slightly larger volume of lumbosacral cerebrospinal fluid than those who underwent less extensive laminectomy and those without a history of lumbar spine surgery. Further studies are warranted to confirm the subgroup analysis findings and elucidate the clinical implications of such differences in the lumbosacral cerebrospinal fluid volume.

Prevalence of temporal bone tegmen defects among patients with Marfan syndrome

Background: Marfan syndrome (MFS) is a genetic disorder affecting connective tissue. The composition of the dura can change. Consequently, lumbo-sacral dural herniations and cerebrospinal fluid (CSF) leaks are encountered, however, they have yet to been described in the temporal bone.Aims/Objectives: To define the prevalence of temporal bone meningocele or encephalocele among patients with MFS.Materials and methods: Reviewed medical records of all adult patients, diagnosed with MFS, who were treated between 1993 and 2018 at a single academic referral institute. Head targeted CT scans were analyzed. The presence of an anterior or lateral skull base defect was recorded.Results: One-hundred and one patients diagnosed with MFS were identified. Twelve of which had suitable CT scans and were enrolled in the study. The median age of patients with defects was 65 years (range 41–71). Five of the twelve patients (41.6%) had tegmen defect. Of the seven defects found, the median size of the defects was 3 mm (range 2–5 mm). All defects were in the temporal bone, none in the anterior skull base.Conclusions and significance: The prevalence of radiological evidence of a temporal bone defect among patients with MFS is high. This is a new, important, and potentially life-threatening association with the syndrome.

Lumbosacral and thoracolumbosacral cerebrospinal fluid volume changes in neonates, infants, children, and adolescents: A retrospective magnetic resonance imaging study.

The volume of cerebrospinal fluid can affect the pharmacokinetics and pharmacodynamics of local anesthetics for spinal anesthesia and other intrathecal medications. The objective of this study was to estimate the lumbosacral cerebrospinal fluid volume and thoracolumbosacral cerebrospinal fluid volume using magnetic resonance images in pediatric patients from neonates and infants to adolescents. Spinal magnetic resonance images of 500 pediatric patients (age <18years) were reviewed. The lumbosacral cerebrospinal fluid volumes of 418 patients and thoracolumbosacral cerebrospinal fluid volumes of 248 patients were measured. The relationship between cerebrospinal fluid volumes and age, height, and weight were evaluated. The lumbosacral and thoracolumbosacral cerebrospinal fluid volumes per weight were calculated to elucidate developmental changes. The lumbosacral and thoracolumbosacral cerebrospinal fluid volumes showed linear correlations with height (r2 =0.730 and r2 =0.661, respectively), whereas they showed curvilinear correlations with age (r2 =0.752 and r2 =0.717, respectively) and weight (r2 =0.734 and r2 =0.734, respectively). The mean lumbosacral cerebrospinal fluid volume per weight (mL/kg) was 0.85 (standard deviation [SD]: 0.19, 95% confidence interval [CI]: 0.81-0.90) in neonates and infants, 0.86 (SD: 0.22, 95% CI: 0.83-0.89) in toddlers and preschoolers, 0.71 (SD: 0.26, 95% CI: 0.66-0.76) in schoolers, and 0.54 (SD: 0.20, 95% CI: 0.49-0.60) in adolescents. The mean thoracolumbosacral cerebrospinal fluid volume per weight (mL/kg) was 1.95 (SD: 0.37, 95% CI: 1.86-2.04) in neonates and infants, 1.82 (SD: 0.41, 95% CI: 1.75-1.88) in toddlers and preschoolers, 1.38 (SD: 0.40, 95% CI: 1.23-1.52) in schoolers, and 0.99 (SD: 0.34, 95% CI: 0.45-1.53) in adolescents. The lumbosacral and thoracolumbosacral cerebrospinal fluid volumes in pediatric patients were much smaller than previously presented values, showing linear correlations with height, and demonstrate curvilinear correlations with age and weight.

Abdominal girth and dorso-sacral distance can be used to estimate lumbosacral cerebral fluid volume.

Patients' abdominal girth and vertebral column length are highly correlated with the spread of local anaesthetics after spinal anaesthesia. Lumbosacral cerebrospinal fluid volume is the primary determinant for spinal spread. Thus, we attempted to verify the hypothesis that abdominal girth and dorso-sacral distance are correlated with lumbosacral cerebrospinal fluid volume. Forty-five healthy volunteers were enrolled in this study to measure lumbosacral cerebrospinal fluid volume using magnetic resonance imaging. The age, height, weight, abdominal girth, dorso-sacral distance and lumbosacral cerebrospinal fluid volume of the volunteers were recorded. Multiple linear regression analysis was used to analyse the correlation between age, height, weight, abdominal girth, dorso-sacral distance and lumbosacral cerebrospinal fluid volume. Two volunteers were excluded because of lumbar disc herniation, leaving 43 volunteers for analysis. Multiple linear regression analysis showed a strong correlation between abdominal girth, dorso-sacral distance and lumbosacral cerebrospinal fluid volume (both P < 0.01). The adjusted R2 was 0.644. Volunteers with small abdominal girth showed clear images of cerebrospinal fluid in the nerve root cuff at the intervertebral foramen in the three-dimensional magnetic resonance imaging reconstruction of lumbosacral cerebrospinal fluid, while the images were vague in volunteers with large abdominal girth. Clearer images implied larger lumbosacral cerebrospinal fluid volume, while vaguer images, smaller lumbosacral cerebrospinal fluid volume. Multiple regression analysis revealed that abdominal girth and dorso-sacral distance were correlated with lumbosacral cerebrospinal fluid volume. Smaller abdominal girths and larger dorso-sacral distances predict larger lumbosacral cerebrospinal fluid volume.

The Effect of Intra-Abdominal Pressure on Sensory Block Level of Single-Shot Spinal Anesthesia for Cesarean Section

(Int J Obstet Anesth. 2015;24(1):35–40) There is an association between pregnancy and increased intrathecal local anesthetic spread (and thus, lower local anesthetic requirements), although the exact mechanisms behind this association are unknown. There are 2 potential anatomic explanations: (1) in the supine position, the pregnant uterus compresses the inferior vena cava (IVC), diverting blood into the vertebral venous system and displacing lumbosacral cerebrospinal fluid (CSF) in a cranial direction; or (2) in addition to direct compression of the IVC, the growing uterus during pregnancy leads to a global increase in intra-abdominal pressure (IAP).

Threshold Selection Criteria for Quantification of Lumbosacral Cerebrospinal Fluid and Root Volumes from MRI

The high variability of CSF volumes partly explains the inconsistency of anesthetic effects, but may also be due to image analysis itself. In this study, criteria for threshold selection are anatomically defined. T2 MR images (n = 7 cases) were analyzed using 3-dimentional software. Maximal-minimal thresholds were selected in standardized blocks of 50 slices of the dural sac ending caudally at the L5-S1 intervertebral space (caudal blocks) and middle L3 (rostral blocks). Maximal CSF thresholds: threshold value was increased until at least one voxel in a CSF area appeared unlabeled and decreased until that voxel was labeled again: this final threshold was selected. Minimal root thresholds: thresholds values that selected cauda equina root area but not adjacent gray voxels in the CSF-root interface were chosen. Significant differences were found between caudal and rostral thresholds. No significant differences were found between expert and nonexpert observers. Average max/min thresholds were around 1.30 but max/min CSF volumes were around 1.15. Great interindividual CSF volume variability was detected (max/min volumes 1.6-2.7). The estimation of a close range of CSF volumes which probably contains the real CSF volume value can be standardized and calculated prior to certain intrathecal procedures.

Lumbar Dural Sac Dimensions Determined by Ultrasound Helps Predict Sensory Block Extent During Combined Spinal-Epidural Analgesia for Labor

Background and Objectives The lumbosacral cerebrospinal fluid volume is a major determinant of the intrathecal spread of local anesthetics. Ultrasound imaging of the lumbar spine allows measurement of dural sac dimensions, which may potentially be used as a surrogate of cerebrospinal fluid volume. The purpose of this study was to investigate the correlation between lumbar dural sac diameter, dural sac length (DSL), and dural sac volume (DSV), measured by ultrasound, and the intrathecal spread of isobaric bupivacaine during combined spinal-epidural (CSE) analgesia for labor. Methods We examined 41 women with singleton pregnancies requesting neuraxial analgesia for labor. Using a 5-2–MHz curved-array ultrasound probe in the paramedian sagittal plane, we measured the dural sac width at each lumbar interspace and the DSL from L1-2 to L5-S1 interspace and calculated the dural sac volume (DSV). Following CSE block with 0.25% isobaric bupivacaine 1.75 mg and fentanyl 15 μg, peak sensory levels (PSLs) were recorded using ice, cotton, and pinprick. Statistical correlation coefficients between dural sac dimensions and PSLs were assessed by Spearman rank correlation. In addition, multiple linear regression models were used to select important predictors of PSLs. Results There was a moderate correlation between DSL and PSL to ice (ρ = −0.62; P Conclusions The length of the lumbar spine determined by ultrasound, rather than the lumbar spine volume, combined with the weight or body mass index of the subject, is of particular value in predicting the intrathecal spread of isobaric bupivacaine during CSE analgesia for labor.

Analysis of serum and cerebrospinal fluid in clinically normal adult miniature donkeys

AIM: To establish reference intervals for serum and cerebrospinal fluid (CSF) parameters in clinically healthy adult miniature donkeys. METHODS: Experiments were conducted on 10 female and 10 male clinically normal adult miniature donkeys, randomly selected from five herds. Lumbosacral CSF collection was performed with the sedated donkey in the standing position. Cell analysis was performed immediately after the samples were collected. Blood samples were obtained from the jugular vein immediately after CSF sample collection. Sodium, potassium, glucose, urea nitrogen, total protein, calcium, chloride, phosphorous and magnesium concentrations were measured in CSF and serum samples. A paired t-test was used to compare mean values between female and male donkeys. RESULTS: The CSF was uniformly clear, colourless and free from flocculent material, with a specific gravity of 1.002. The range of total nucleated cell counts was 2–4 cells/μL. The differential white cell count comprised only small lymphocytes. No erythrocytes or polymorphonuclear cells were observed on cytological examination. Reference values were obtained for biochemical analysis of serum and CSF. Gender had no effect on any variables measured in serum or CSF (p>0.05). CONCLUSION AND CLINICAL RELEVANCE: CSF analysis can provide important information in addition to that gained by clinical examination. CSF analysis has not previously been performed in miniature donkeys; this is the first report on the subject. In the present study, reference intervals for total nucleated cell count, total protein, glucose, urea nitrogen, sodium, potassium, chloride, calcium, phosphorous and magnesium concentrations of serum and CSF were determined for male and female miniature donkeys.

CARACTERÍSTICAS FÍSICO-QUÍMICAS E CITOLÓGICAS DO LÍQUOR DE OVINOS SADIOS DA RAÇA SANTA INÊS

The occurrence of neurological disease is widespread in clinical medicine of domestic animal, but there are few studies documenting the normal composition of cerebrospinal fluid (CSF) in ovine, especially Santa Ines breed. This study establishes the parameters for normal physiochemical and cytological analysis of cerebrospinal fluid as an aid for diagnosis of neurological disorders in this breed. For this, 40 adult ovine animals, aged between six and 18 months, were used for puncture of lumbosacral CSF. The aspect and color shower a clear and colorless pattern. The average density was 1.006 ± 0.0064, protein concentration was 25.27 ± 6.64 mg/dL and glucose concentration was 46.89 ± 10.99 mg/dL. Enzymes had concentration averages of 116.72 ± 80.79 U/L for creatine kinase; 17.43 ± 11.91 U/L for lactate dehydrogenase and 17.08 ± 3.85 U/L for aspartate aminotransferase. The results for the global cell count were 4.61 ± 4.96 leukocytes/µL (0 to 15 lymphocytes, 0 to 10 monocytes, 1 to 14 neutrophils). Considering the lack of reference data for the physiochemical and cytological CSF analysis of Santa Ines ovine breed, it is recommended that the results obtained in this study are considered normal values for the breed.KEYWORDS: biochemistry; CFS; cytology; small ruminant; standard features.

Lumbar Dural Sac Dimensions Determined by Ultrasound Helps Predict Sensory Block Extent During Combined Spinal-Epidural Analgesia for Labor

The lumbosacral cerebrospinal fluid volume is a major determinant of the intrathecal spread of local anesthetics. Ultrasound imaging of the lumbar spine allows measurement of dural sac dimensions, which may potentially be used as a surrogate of cerebrospinal fluid volume. The purpose of this study was to investigate the correlation between lumbar dural sac diameter, dural sac length (DSL), and dural sac volume (DSV), measured by ultrasound, and the intrathecal spread of isobaric bupivacaine during combined spinal-epidural (CSE) analgesia for labor. We examined 41 women with singleton pregnancies requesting neuraxial analgesia for labor. Using a 5-2-MHz curved-array ultrasound probe in the paramedian sagittal plane, we measured the dural sac width at each lumbar interspace and the DSL from L1-2 to L5-S1 interspace and calculated the dural sac volume (DSV). Following CSE block with 0.25% isobaric bupivacaine 1.75 mg and fentanyl 15 μg, peak sensory levels (PSLs) were recorded using ice, cotton, and pinprick. Statistical correlation coefficients between dural sac dimensions and PSLs were assessed by Spearman rank correlation. In addition, multiple linear regression models were used to select important predictors of PSLs. There was a moderate correlation between DSL and PSL to ice (ρ = -0.62; P < 0.0005) and to pinprick (ρ = -0.52; P = 0.017). Similarly, there was a moderate correlation between DSV and PSL to ice (ρ = -0.56; P = 0.004) and to pinprick (ρ = -0.61; P < 0.0008). Neither the DSL nor DSV correlated with PSL to cotton. Multiple linear regression analysis revealed that DSL, weight, and body mass index contributed to PSLs. The length of the lumbar spine determined by ultrasound, rather than the lumbar spine volume, combined with the weight or body mass index of the subject, is of particular value in predicting the intrathecal spread of isobaric bupivacaine during CSE analgesia for labor.

The correlation between cauda equina nerve root volume and sensory block height after spinal anaesthesia with glucose-free bupivacaine

We examined the association between cauda equina nerve root volume and sensory block height in 15 patients undergoing spinal anaesthesia with 0.5% glucose-free bupivacaine. Magnetic resonance imaging and a separate image segmentation program were used to calculate the volume of the nerve roots. Nerve root volume was also correlated with lumbosacral cerebrospinal fluid volume and with patients' physical characteristics. Nerve root volume correlated negatively with sensory block height (Spearman rho -0.61 (95% CI -0.85 to -0.14)) and body mass index (Spearman rho -0.66 (95% CI -0.87 to -0.24)) but positively with cerebrospinal fluid volume (Spearman rho 0.76 (95% CI 0.43-0.91)). Factors that are thought to influence cerebrospinal fluid volume, such as body mass index, might similarly affect the volume of the nerve roots. The size of the nerve roots may influence the spread of spinal anaesthesia.

Distribution of Epidural Saline Upon Injection and the Epidural Volume Effect in Pregnant Women

How injected epidural solution is distributed and affects the epidural volume in pregnant women are unclear. Lumbar epidural catheters were placed using the loss-of-resistance technique with saline in eight full-term (39 weeks' gestation) parturients for labor and eight volunteer nonpregnant women. Lumbosacral cerebrospinal fluid volume was measured on thoracic and lumbosacral axial magnetic resonance images. Another image series was obtained after injecting 10 ml saline into the epidural space through the catheter to compare the saline distribution (dural sac coating and exit from foramina) and cerebrospinal fluid volume before and after epidural injection. Dural sac coating was based on observation of epidural saline in the anterior epidural space after injection in axial magnetic resonance images at the pedicle levels from T12 to L5. Saline leakage from the foramina was determined by the same method at six disc levels from T11-T12 to L4-L5. Significantly fewer images of pregnant women than nonpregnant women showed saline surrounding the dural sac (0 [0-0] vs. 3 [1-4], median [interquartile range]; P < 0.01) and saline leakage from the foramina (0 [0-1] vs. 6 [4-6]; P < 0.01). The mean reduction in cerebrospinal fluid volume was significantly greater in pregnant (8.4 ± 1.4 ml; mean ± SD) than in nonpregnant women (4.6 ± 1.1 ml; P < 0.001). Limited dural sac coating and decreased leakage from the foramina of saline injected into the epidural space may account for the facilitation of longitudinal spread of epidural analgesia in pregnant women. The epidural volume effect is greater in pregnant than in nonpregnant women.

Spinal cerebrospinal fluid volume in healthy elderly individuals

The amount of spinal cerebrospinal fluid (CSF) could be of importance for the understanding of CSF dynamics, CSF biomarker analyses as well as for the amount and effect of anaesthesia using intrathecally administered drugs. However, knowledge of spinal CSF volumes is scarce. The main purpose of this article is to present data on spinal CSF volumes. In total, 22 healthy individuals aged between 64 and 76 years underwent MR imaging with a 3D balanced turbo field echo pulse sequence, which provided high contrast between spinal cord, CSF and the extradural surroundings. The entire spinal CSF volume, the cervical, thoracic, and lumbosacral CSF volumes and the spinal cord volume were calculated. The total spinal CSF volume was 81 ± 13 ml (range 52-103 ml). The amount of CSF in the cervical region was 19 ± 4 ml, in the thoracic region 38 ± 8 and in the lumbosacral region 25 ± 7 ml. There was no difference between genders nor was there any correlation with height. The volume of the spinal cord was 20 ± 3 ml. The results present new magnetic resonance imaging-based data on the spinal CSF volume in healthy elderly individuals.

Gestation-related Reduction in Lumbar Cerebrospinal Fluid Volume and Dural Sac Surface Area

Onuki, E.; Higuchi, H.; Takagi, S.; Nishijima, K.; Fujita, N.; Matsuura, T.; Ozaki, M. Author Information

Cervical vertebral canal endoscopy in the horse: Intra- and post operative observations

Despite modern medical diagnostic imaging, it is not possible to identify reliably the exact location of spinal cord compression in horses with cervical vertebral stenotic myelopathy (CVSM). Vertebral canal endoscopy has been successfully used in man and a technique for cervical vertebral canal endoscopy (CVCE) has been described in equine cadavers. To determine the feasibility and safety of CVCE in healthy mature horses. Six healthy mature horses were anaesthetised. A flexible videoendoscope was subsequently introduced via the atlanto-occipital space into the epidural space (epiduroscopy, Horses 1-3) or the subarachnoid space (myeloscopy, Horses 4-6) and advanced to the 8th cervical nerve. Neurological examinations were performed after surgery and lumbosacral cerebrospinal fluid (CSF) analysed in horses that had undergone myeloscopy. All procedures were completed successfully and all horses recovered from anaesthesia. Anatomical structures in the epidural space (including the dura mater, nerve roots, fat and blood vessels) and subarachnoid space (including the spinal cord, blood vessels, arachnoid trabeculations, nerve roots and the external branch of the accessory nerve) were identified. During epiduroscopy, a significant increase in mean arterial pressure was recognised, when repeated injections of electrolyte solution into the epidural space were performed. In one horse of the myeloscopy group, subarachnoid haemorrhage and air occurred, resulting in transient post operative ataxia and muscle fasciculations. No complications during or after myeloscopy were observed in the other horses. CSF analysis indicated mild inflammation on Day 7 with values approaching normal 21 days after surgery. Endoscopic examination of the epidural and subarachnoid space from the atlanto-occipital space to the 8th cervical nerve is possible and can be safely performed in healthy horses. Cervical vertebral canal endoscopy might allow accurate identification of the compression site in horses with CVSM and aid diagnosis of other lesions within the cervical vertebral canal.

Gestation-Related Reduction in Lumbar Cerebrospinal Fluid Volume and Dural Sac Surface Area

Facilitation of the spread of neuraxial anesthesia in pregnant women may be attributable in part to compression of the dural sac by the engorged epidural venous plexus. In this study, we used magnetic resonance imaging to examine pregnancy-induced changes in the lumbosacral cerebrospinal fluid (CSF) volume and dural sac surface area. Magnetic resonance images of 18 healthy women (mean age 29 yr, mean height 158 cm, and mean weight 58 kg) were obtained to measure lumbosacral CSF volume and dural sac surface area in the nonpregnant and pregnant states (median 36 wk gestation [31-39]) and the paired images were compared. The mean lumbosacral CSF volume and dural sac surface area in the nonpregnant state were 39.6 +/- 5.8 mL and 11.0 +/- 0.8 cm(2), respectively. Pregnancy was associated with compression of the dural sac, resulting in a significantly reduced mean CSF volume (33.2 +/- 6.2 mL) and dural sac surface area (9.9 +/- 1.0 cm(2)) in all subjects (P < 0.001). The mean change in CSF volume and dural sac surface area was 16.7% +/- 0.8% and 10.0% +/- 0.5%, respectively. Gestational week (between 31 and 39 wk) correlated significantly with the reduction in CSF volume (rho = 0.74, P < 0.001) and dural sac surface area (rho = 0.66, P < 0.01). These findings indicate an association between gestational week (Weeks 31-39) and a reduction in both CSF volume and dural sac surface area. These reductions may, at least in part, explain the facilitation of the spread of intrathecal anesthesia in pregnant women.

Evidence that intrathecal morphine-3-glucuronide may cause pain enhancement via toll-like receptor 4/MD-2 and interleukin-1β

Morphine-3-glucoronide (M3G) is a major morphine metabolite detected in cerebrospinal fluid of humans receiving systemic morphine. M3G has little-to-no affinity for opioid receptors and induces pain by unknown mechanisms. The pain-enhancing effects of M3G have been proposed to significantly and progressively oppose morphine analgesia as metabolism ensues. We have recently documented that morphine activates toll-like receptor 4 (TLR4), beyond its classical actions on μ-opioid receptors. This suggests that M3G may similarly activate TLR4. This activation could provide a novel mechanism for M3G-mediated pain enhancement, as (a) TLR4 is predominantly expressed by microglia in spinal cord and (b) TLR4 activation releases pain-enhancing substances, including interleukin-1 (IL-1). We present in vitro evidence that M3G activates TLR4, an effect blocked by TLR4 inhibitors, and that M3G activates microglia to produce IL-1. In vivo, intrathecal M3G (0.75 μg) induced potent allodynia and hyperalgesia, blocked or reversed by interleukin-1 receptor antagonist, minocycline (microglial inhibitor), and (+)-and (−)-naloxone. This latter study extends our prior demonstrations that TLR4 signaling is inhibited by naloxone nonstereoselectively. These results with (+)-and (−)-naloxone also demonstrate that the effects cannot be accounted for by actions at classical, stereoselective opioid receptors. Hyperalgesia (allodynia was not tested) and in vitro M3G-induced TLR4 signaling were both blocked by 17-DMAG, an inhibitor of heat shock protein 90 (HSP90) that can contribute to TLR4 signaling. Providing further evidence of proinflammatory activation, M3G upregulated TLR4 and CD11b (microglial/macrophage activation marker) mRNAs in dorsal spinal cord as well as IL-1 protein in the lumbosacral cerebrospinal fluid. Finally, in silico and in vivo data support that the glucuronic acid moiety is capable of inducing TLR4/MD-2 activation and enhanced pain. These data provide the first evidence for a TLR4 and IL-1 mediated component to M3G-induced effects, likely of at least microglial origin.

Immunogenicity of intrathecal plasmid gene delivery: cytokine release and effects on transgene expression

One method for the delivery of therapeutic proteins to the spinal cord is to inject nonviral gene vectors including plasmid DNA into the cerebrospinal fluid (CSF) that surrounds the spinal cord (intrathecal space). This approach has produced therapeutic benefits in animal models of disease and several months of protein expression; however, there is little information available on the immune response to these treatments in the intrathecal space, the relevance of plasmid CpG sequences to any plasmid-induced immune response, or the effect of this immune response on transgene expression. In the present study, coding or noncoding plasmids were delivered to the intrathecal space of the lumbar spinal region in rats. Lumbosacral CSF was then collected at various time points afterwards for monitoring of cytokines and transgene expression. This work demonstrates, for the first time, increased tumor necrosis factor-alpha and interleukin-1 in response to intrathecal plasmid vector injection and provides evidence indicating that this response is largely absent in a CpG-depleted vector. Transgene expression in the CSF is not significantly affected by this immune response. Expression after intrathecal plasmid injection is variable across rats but correlates with the amount of tissue associated plasmid and is increased by disrupting normal CSF flow. The data obtained in the present study indicate that plasmid immunogenicity may affect intrathecal plasmid gene therapy safety but not transgene expression in the CSF. Furthermore, the development of methods to prevent loss of plasmid via CSF flow out of the central nervous system through the injection hole and/or natural outflow routes may increase intrathecal plasmid gene delivery efficacy.

Intrathecal Injection of Naked Plasmid DNA Provides Long-term Expression of Secreted Proteins

Therapeutic benefit has been reported to result from intrathecal (i.t.) injection of transgene vectors, including naked DNA. However, most studies using naked DNA have measured only the transgene expression of intracellular proteins. Here we demonstrate that i.t. injection of naked DNA can result in long-term expression of secreted proteins. Plasmids expressing either secreted alkaline phosphatase (SEAP) or human interleukin-10 (hIL-10) were injected into the i.t. space in rats, and transgene products were repeatedly measured in the cerebrospinal fluid (CSF). Both SEAP and hIL-10 were maximal at 1 and 2 days after the injection and still detectable at 4 months. The utilization of a plasmid having two features that are hypothesized to increase gene expression (matrix attachment regions (MARs) and lack of CpG dinucleotides) resulted in a significant increase in gene expression. Reinjection of SEAP or hIL-10 plasmids after 4 months significantly increased protein levels at 1 and 14 days after the reinjection. SEAP was uniformly distributed between the DNA delivery site (approximately vertebral level T13) and the lumbar puncture site (L5/L6 inter-vertebral space), was reduced at the cisterna magna, and was detectable, though at much lower levels, in serum. These data suggest that naked DNA has the potential to be used as a therapeutic tool for applications that require long-term release of transgenes into the CSF.