Matrix metalloprotease-9 activity in the cerebrospinal fluid and spinal injury severity in dogs with intervertebral disc herniation

We investigated whether matrix metalloproteinase (MMP)-9 expression in the cerebrospinal fluid (CSF) of dogs with intervertebral disc herniation (IVDH) is associated with the severity of neurological signs and prognosis. CSF from the cisterna magna (C-CSF) and the lumbar spine (L-CSF) of 34 dogs with IVDH was analyzed using zymography. Activity of MMP-9 in L-CSF was detected in 6 of 34 dogs with IVDH, often for more than 7 days after injury. MMP-9 activity was not detected from any of the C-CSF samples. Of the six cases that were MMP-9 positive, all four cases with grade V that had loss of deep pain were non-ambulatory 6 months after treatment. The remaining two cases with grade III and IV could recover mobility. In dogs with grade V thoracolumbar IVDH, MMP-9 expression in the CSF may indicate severe spinal cord injury with poor prognosis.     

Gelatinase activity in synovial fluid and synovium obtained from healthy and osteoarthritic joints of dogs

OBJECTIVE: To determine matrix metalloproteinase (MMP) activity in synovial fluid (SF) obtained from the joints of dogs with degenerative joint disease (DJD) secondary to various underlying conditions. SAMPLE POPULATION: 35 samples of SF obtained from 18 clinically normal (control) dogs and 34 samples of SF obtained from 17 dogs with DJD; dogs with DJD were from 2 populations (client-owned dogs and research dogs that had DJD secondary to the lysosomal storage disease mucopolysaccharidosis VII). PROCEDURE: MMP activity in samples of SF was semiquantitatively examined by use of gelatin or casein zymography. Western blot analysis was performed by use of antibodies for MMP-2 and MMP-9. In addition, in situ MMP activity was observed in sections of synovial membrane obtained from healthy and osteoarthritic joints. RESULTS: Samples of SF from osteoarthritic joints had higher MMP-2 activity and dramatically increased MMP-9 activity, compared with values for healthy joints. Substrate-overlay analyses indicated minimal gelatin-degrading activity in synoviocytes obtained from control dogs, whereas greater activity was seen in osteoarthritic synoviocytes, with additional activity in the underlying tissue. CONCLUSIONS AND CLINICAL RELEVANCE: Higher MMP-2 activity and dramatic increases in MMP-9 activity were associated with the osteoarthritic state, even though MMP-2 activity was detected in healthy joints. This study expands information on MMP production in SF of osteoarthritic joints in other species and documents the similarity of MMP activity patterns regardless of the cause of DJD.     

Matrix metalloprotease-9 activity in the cerebrospinal fluid and spinal injury severity in dogs with intervertebral disc herniation

We investigated whether matrix metalloproteinase (MMP)-9 expression in the cerebrospinal fluid (CSF) of dogs with intervertebral disc herniation (IVDH) is associated with the severity of neurological signs and prognosis. CSF from the cisterna magna (C-CSF) and the lumbar spine (L-CSF) of 34 dogs with IVDH was analyzed using zymography. Activity of MMP-9 in L-CSF was detected in 6 of 34 dogs with IVDH, often for more than 7 days after injury. MMP-9 activity was not detected from any of the C-CSF samples. Of the six cases that were MMP-9 positive, all four cases with grade V that had loss of deep pain were non-ambulatory 6 months after treatment. The remaining two cases with grade III and IV could recover mobility. In dogs with grade V thoracolumbar IVDH, MMP-9 expression in the CSF may indicate severe spinal cord injury with poor prognosis.     

Association of cerebrospinal fluid analysis findings with clinical signs and outcome in acute nonambulatory thoracolumbar disc disease in dogs

Background: Cerebrospinal fluid (CSF) pleocytosis recently was associated with the severity of neurologic signs in dogs with intervertebral disc disease (IVDD). Hypothesis/Objectives: To look for an association among CSF cell counts, total protein concentration, and severity of neurologic signs at presentation with outcome in dogs with acute thoracolumbar IVDD. Our hypothesis was that CSF total nucleated cell count (TNCC) and percentage cell types would be associated with the severity of spinal cord damage and therefore with both the presenting clinical signs and the prognosis of affected dogs. Animals: Fifty‐four dogs with acute nonambulatory thoracolumbar IVDD were evaluated. Methods: Retrospective study. Signalment, neurologic grade, CSF TNCC, protein concentration, red blood cells count and differential cell percentages, and short‐ and long‐term outcomes were evaluated. Results: CSF pleocytosis (>5 cells/μL) was present in 54% of dogs and was positively associated with neurologic grade at presentation and with postoperative time to regaining ambulation. Neutrophils were observed most frequently. The percentage of CSF macrophages and macrophage to monocyte ratio were higher (P= .001, for both) in dogs presented without deep pain sensation (DPS) that did not regain ambulation. Receiver operator characteristics curve analysis yielded a cut‐off point of 13% macrophages with a sensitivity and specificity of 100 and 83%, respectively, for prediction of a negative outcome. Conclusions and Clinical Importance: CSF pleocytosis is positively associated with the severity of spinal cord damage in dogs with thoracolumbar IVDD. The percentage of CSF macrophages can be used as a prognostic indicator for regaining ambulation in dogs that have lost DPS.     

Canine cerebral leishmaniasis: Potential role of matrix metalloproteinase-2 in the development of neurological disease

Matrix metalloproteinases (MMPs) are a group of calcium- and zinc-dependent endopeptidases that are involved in maintaining the extracellular matrix. MMP-2 and MMP-9 are thought to be related to the disruption of the blood-brain-barrier (BBB) by their ability to cleave type IV collagen, the main component of the basal membrane. To establish the presence of MMP-2 and MMP-9 in the pathogenesis of canine cerebral leishmaniasis, we examined the levels of these metalloproteinases in the cerebrospinal fluid (CSF) and serum of dogs with visceral leishmaniasis and neurological symptoms (n=16) and in the CSF and serum of uninfected healthy dogs (n=10) using zymography. In the CSF of dogs with cerebral leishmaniasis there was a massive presence of active MMP-2, whereas only the levels of both proMMP-2 and proMMP-9 were elevated in the serum. Although the detected MMP activity in the CSF might merely be related to CNS inflammation, these enzymes may also play a collaborative role in the disease progression. Both MMP-2 and MMP-9 are known to target critical constituents of the BBB, and once activated, they may promote cerebral barrier breakdown, allowing the entrance of inflammatory cells and proteins within the nervous system milieu.     

Evaluation of the association between spondylosis deformans and clinical signs of intervertebral disk disease in dogs: 172 cases (1999-2000)

OBJECTIVE: To evaluate the association between spondylosis deformans and clinical signs of intervertebral disk disease (IVDD) in dogs. DESIGN: Retrospective case series. ANIMALS: 210 dogs. PROCEDURE: Records of 172 dogs with clinical signs of IVDD and 38 dogs with other neurologic disorders were reviewed. Signalment, sites of spondylosis, severity of associated osteophytosis, type of disk herniation, and duration of signs were recorded. RESULTS: Dogs with IVDD had significantly fewer sites of involvement and lower grades of spondylosis deformans, compared with those in the non-IVDD group. When groups were adjusted for age and weight via multivariate linear regression, there were no differences in severity of osteophytosis or number of affected sites. Dogs with type II disk disease had higher numbers of affected sites and more severe changes, compared with dogs with type I disk herniation. There was no difference between groups in the rate at which IVDD was diagnosed at sites of spondylosis, compared with the rate at which IVDD was diagnosed in unaffected disk spaces. Areas of spondylosis were closer to sites of IVDD that elicited clinical signs than to randomly chosen intervertebral spaces, and distances between sites of spondylosis and sites of IVDD had a bimodal appearance. CONCLUSIONS AND CLINICAL RELEVANCE: An association may exist between radiographically apparent spondylosis and type II disk disease; type I disk disease was not associated with spondylosis. Spondylosis in radiographs of dogs with suspected type I disk disease is not clinically important. Spatial associations among sites of spondylosis and sites of IVDD may be coincidental or associated with vertebral column biomechanics.     

Beta-2-microglobulin levels in the cerebrospinal fluid of normal dogs and dogs with neurological disease

BACKGROUND: Cerebrospinal fluid (CSF) analysis is the basis for establishing a diagnosis of central nervous system (CNS) inflammation. However, the information provided by routine CSF analysis is limited. Determination of CSF beta-2-microglobulin (beta2m) concentration has been used diagnostically in humans to identify inflammatory CNS disease; we hypothesized that it may have similar value in dogs. OBJECTIVES: The objective of this study was to measure (beta2m concentration in the CSF of clinically healthy dogs and compare the values to those observed in dogs with inflammatory CNS disease and intervertebral disc disease (IVDD). METHODS: CSF was collected from 10 clinically healthy laboratory dogs and 11 dogs each with inflammatory CNS disease and IVDD. Routine CSF analysis was performed, and (beta2m concentration was measured by ELISA. CSF (beta2m concentration and CSF:serum (beta2m ratio were compared between groups by ANOVA. Linear relationships between CSF total nucleated cell count (TNCC), RBC count, total protein concentration, and (beta2m concentration were assessed by regression analysis. RESULTS: The mean (+/- SD) CSF (beta2m concentration in clinically healthy dogs was 0.36 (+/- 0.05 microg/mL (cisternal) and 0.40 (+/- 0.07 microg/mL (lumbar). Median CSF (beta2m concentration in dogs with IVDD (0.46 microg/mL) and inflammatory CNS disease (0.85 microg/mL) differed from that of controls (0.36 microg/mL; P=.002). The concentration also differed between the 2 disease groups (P=.01). Five dogs with inflammatory CNS disease had CSF:serum (beta2m ratios >1. A correlation was identified between TNCC and (beta2m concentration (r=0.69, P=.0003). CONCLUSIONS: CSF (beta2m concentration is higher in dogs with IVDD and inflammatory CNS disease, with highest values seen with inflammatory disease. This may be attributed in part to the correlation between CSF (beta2m concentration and TNCC, but also may reflect intrathecal immune activation.     

Analysis of cartilage oligomeric matrix protein and matrix metalloproteinase-9 in cerebrospinal fluid of miniature dachshund with intervertebral disc herniation

We evaluated whether the cerebrospinal fluid (CSF) concentration of cartilage oligomeric matrix protein (COMP) is related to disease severity, prognosis and matrix metalloproteinase (MMP)-9 activity of the CSF in miniature dachshund with intervertebral disc herniation. Samples were obtained from 23 patients and 6 normal dogs, and all patients received hemilaminectomy. Twenty dogs recovered successfully and 3 of 11 dogs without deep nociception had MMP-9 activity in the CSF and an unsuccessful outcome. The COMP levels from patients were significantly higher than those from normal dogs. MMP-9 activity and neurological severity were not related to the COMP levels. However, the COMP levels from 3 unsuccessful cases that had MMP-9 activity were significantly lower than those from all recovered cases and/or successful cases without deep nociception. Concerning severe cases, increased proteolytic activity might affect the COMP concentration and prognosis due to MMP-9 associated deleterious effects.     

Analysis of cartilage oligomeric matrix protein and matrix metalloproteinase-9 in cerebrospinal fluid of miniature dachshund with intervertebral disc herniation

We evaluated whether the cerebrospinal fluid (CSF) concentration of cartilage oligomeric matrix protein (COMP) is related to disease severity, prognosis and matrix metalloproteinase (MMP)-9 activity of the CSF in miniature dachshund with intervertebral disc herniation. Samples were obtained from 23 patients and 6 normal dogs, and all patients received hemilaminectomy. Twenty dogs recovered successfully and 3 of 11 dogs without deep nociception had MMP-9 activity in the CSF and an unsuccessful outcome. The COMP levels from patients were significantly higher than those from normal dogs. MMP-9 activity and neurological severity were not related to the COMP levels. However, the COMP levels from 3 unsuccessful cases that had MMP-9 activity were significantly lower than those from all recovered cases and/or successful cases without deep nociception. Concerning severe cases, increased proteolytic activity might affect the COMP concentration and prognosis due to MMP-9 associated deleterious effects.     

Characteristics of dogs admitted for treatment of cervical intervertebral disk disease: 105 cases (1972-1982).

Case histories of 105 dogs that were treated for cervical intervertebral disk disease (IVDD) were studied retrospectively. To compare with previous data, dogs were grouped by age, gender, and breed. Dogs were also grouped by clinical signs of disease, and by presence and location of radiologic change. The age range of cases of cervical IVDD was 1 to 13 years (mean, 6.3 years). Fifty-nine percent of dogs treated for cervical IVDD were females, but the proportion of diseased females was similar to females in total hospital admissions. Twenty-eight breeds of dogs were treated for cervical IVDD. Dachshunds and Beagles were significantly over represented (P less than or equal to 0.001). However, gender-breed interaction was not observed. Prevalence of radiologic evidence of disk disease was detected at the following levels of the vertebral column: C2-3, 29%; C3-4, 24%; C4-5, 21%; C5-6, 15%; C6-7, 9%; and C7-T1, 2%. Significant difference was not observed in prevalence of cervical IVDD affecting the first 4 disk spaces. However, prevalence of cervical IVDD at C7-T1 was significantly less than that involving the first 4 disk spaces (P less than 0.02), and the space at C6-7 was significantly less affected than were the first 3 spaces (P less than 0.08). Significant association was not evident between clinical signs (pain and neurologic deficits) and radiologic signs of IVDD, although neurologic deficits were more likely to be observed in association with radiologic signs than with signs of pain.     

Characterization of matrix metalloproteinase-2 and -9 in cerebrospinal fluid of clinically normal dogs

OBJECTIVE: To characterize matrix metalloproteinase (MMP)-2 and -9 in CSF of clinically normal dogs. SAMPLE POPULATION: Samples of CSF collected from 23 dogs. PROCEDURE: Dogs were anesthetized, CSF samples were collected, and dogs were then euthanatized. Each CSF sample was evaluated immediately for RBC count, WBC count, and protein and glucose concentrations, and cytologic examination also was performed. Samples were considered normal when protein concentration was < 25 mg/dL and CSF contained < 6WBCs/microL and < 25 RBCs/microL. Samples were stored at -70 degrees C. Sections of brain tissue were collected and processed for histologic examination. The MMPs were evaluated by use of gelatin zymography and a polyclonal antibody-based sandwich ELISA. RESULTS: Mean WBC count for CSF samples was < 1 WBC/microL (range, 0 to 3 WBCs/mL). Mean protein concentration was 12 mg/dL (range, 8 to 17 mg/dL). Mean RBC count was 3.65 RBCs/microL (range, 0 to 21 RBCs/microL). All CSF samples generated a clear band on zymography gels that corresponded to the human commercial standard of proenzyme MMP-2. Other major clear bands were not detected on zymography gels. Bands correlating to MMP-9 were not detected in any samples. The ELISA results revealed a mean +/- SD proenzyme MMP-2 concentration of 5.61 +/- 1.92 ng/mL (range, 3.36 to 10.83 ng/mL). CONCLUSIONS AND CLINICAL RELEVANCE: The proenzyme form of MMP-2 is detectable in CSF of clinically normal dogs, whereas MMP-9 is not detectable. Additional investigation of MMPs in CSF from dogs with various diseases of the nervous system is indicated.     

Matrix metalloproteinase activity in stifle synovial fluid of cranial cruciate ligament deficient dogs and effect of postoperative doxycycline treatment

This prospective clinical study investigated the activity of matrix metalloproteinases (MMPs) in stifle synovial fluid (SF) of 13 dogs with acute cranial cruciate ligament (CCL) rupture, and the effect of a postoperative doxycycline treatment. MMP-2, 3, 9 and 13 activities were compared with respect to the time of sampling (preoperatively or 1 month after surgical stabilisation) and the type of postoperative adjuvant treatment (doxycycline or not). No significant activity was detected for both MMP-3 and MMP-13. MMP-2 and MMP-9 activities were found to be significantly highly increased in SF of CCL ruptured stifles compared to control stifles of unaffected dogs. No significant effect from surgical stabilisation and postoperative doxycycline treatment on MMP-2 and MMP-9 activities was found, indicating that doxycycline may not be an appropriate postoperative medical treatment after CCL rupture.     

Cerebrospinal fluid analysis and magnetic resonance imaging in the diagnosis of neurologic disease in dogs: a retrospective study

BACKGROUND: Diagnosis of central nervous system (CNS) abnormalities in dogs can be challenging antemortem. Historically, cerebrospinal fluid (CSF) analysis has been used for routine diagnostic evaluation of animals with suspected neurologic disease; however, with increasing availability of magnetic resonance (MR) imaging, the need for concurrent CSF analysis may be questioned. OBJECTIVE: The purpose of this study was to retrospectively assess and compare the diagnostic information contributed from MR imaging and CSF analysis in a population of dogs presenting with neurologic disease. METHODS: Results of concurrent MR imaging and CSF analysis were evaluated in dogs presented for neurologic diseases. Based on clinical diagnosis, the sensitivity of CSF analysis and MR imaging for detecting a nervous system abnormality was calculated. Dogs with diagnoses confirmed by other diagnostic modalities were also evaluated separately. RESULTS: A total of 256 dogs were included in the study. For clinical diagnoses in which abnormalities were expected, MR imaging abnormalities were found in 89% and CSF abnormalities in 75% of dogs; CSF abnormalities were more common than MR imaging abnormalities only in inflammatory CNS disease. The majority of CSF abnormalities were nonspecific; an etiologic diagnosis was determined in only 2% of CSF samples. MR imaging excelled in detecting structural disorders, revealing 98% of vertebral abnormalities. In confirmed cases (n = 55), 76% of MR images and 9% of CSF samples were diagnostic. When intervertebral disk disease (IVDD) and vertebral malformation were excluded from analysis (n = 16 remaining), 25% of MR images and 6% of CSF cytology results were highly indicative of the confirmed diagnoses; CSF titer results provided the diagnosis in 25% of these cases. CONCLUSION: CSF analysis may not be necessary when MR findings of IVDD or vertebral malformation/instability are obvious; however, when the cause of neurologic disorder is uncertain, concurrent MR imaging and CSF analysis provides the greatest assistance in establishing a clinical diagnosis.     

Magnetic resonance imaging findings in dogs with thoracolumbar intervertebral disk disease: 69 cases (1997-2005)

OBJECTIVE: To determine magnetic resonance imaging (MRI) abnormalities in dogs with intervertebral disk disease (IVDD) and develop a classification scheme for IVDD in dogs based on MRI findings. DESIGN: Retrospective case series. ANIMALS: 69 dogs. PROCEDURE: Medical records of dogs admitted because of thoracolumbar IVDD in which MRI of T9 through L7 had been performed were reviewed. RESULTS: A total of 759 intervertebral disk spaces were examined. Of these, 342 (45.1%) were classified as having a normal MRI appearance; the remaining 417 (54.9%) had various types of IVDD. Disk degeneration was identified in 276 disk spaces in 56 dogs, bulging of the intervertebral disk was identified in 37 disk spaces in 24 dogs, disk protrusion was identified in 54 disk spaces in 32 dogs, and disk extrusion was identified in 50 disk spaces in 48 dogs. Cartilage endplate changes were identified in 35 vertebrae in 17 dogs, and increased signal intensity of the spinal cord was identified in 21 dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Four types of IVDD (disk degeneration, bulging of the intervertebral disk, disk protrusion, and disk extrusion) were identified on the basis of MRI findings in dogs with thoracolumbar IVDD. We recommend that a standardized nomenclature be adopted for the various types of thoracolumbar IVDD in dogs.     

Frequency of urinary tract infection in catheterized dogs and comparison of bacterial culture and susceptibility testing results for catheterized and noncatheterized dogs with urinary tract infections

OBJECTIVE: To determine frequency of urinary tract infections (UTIs) in catheterized dogs that had intervertebral disk disease (IVDD) or disease other than IVDD and compare bacterial culture and susceptibility testing results for catheterized and noncatheterized dogs with UTIs. DESIGN: Retrospective cohort study. ANIMALS: 147 catheterized dogs (105 with IVDD and 42 with other diseases) and 99 noncatheterized dogs with UTIs. PROCEDURES: Medical records were reviewed for signalment, history, clinical problem, duration of urinary tract catheterization, administration of drugs, and urine bacterial culture and susceptibility testing results. RESULTS: Forty-two percent (44/105) of dogs with IVDD and 55% (23/42) of dogs with other diseases had UTIs; this difference was not significant. For catheterized dogs, the odds of UTI were increased by 20% for each year increase in age, 27% for each day increase in duration of catheterization, and 454% with antimicrobial administration. Escherichia coli and Proteus spp were more frequently isolated from noncatheterized dogs, whereas Enterobacter spp and Staphylococcus spp were more frequently isolated from catheterized dogs. There was no significant difference in frequency of 1, 2, or 3 isolates between groups. Proportions of antimicrobials to which the most frequently isolated bacteria were resistant were not significantly different between groups. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that urinary tract catheterization is a reasonable alternative for management of dogs with urinary bladder dysfunction, but that duration of catheterization should be minimized and indiscriminate antimicrobial administration to dogs with indwelling urinary catheters should be avoided.     

Matrix metalloproteinase-9 in laminae of black walnut extract treated horses correlates with neutrophil abundance

We sought to determine whether a correlation exists between neutrophil infiltration and tissue matrix metalloproteinase-9 (MMP-9) content in digital laminae collected during the prodromal and acute phases of laminitis in horses treated with an aqueous black walnut heartwood extract (BWE). Hoof laminar tissue was obtained at the onset of leukopenia and at the onset of clinical signs of lameness from BWE-treated horses and at equivalent times from control horses. Thin sections of laminae were screened for neutrophils by immunohistochemistry with an anti-CD13 monoclonal antibody and extracts of the same tissues were screened for SDS-renaturable and native MMP-9 activities by denaturing and non-denaturing gelatin zymography. Samples were also screened for MMP-2 and MMP-9 gene expression by RT-qPCR. Control laminae were devoid of both MMP-9 and neutrophils, whereas neutrophils and SDS-renaturable MMP-9 activity were detected in laminae from BWE-treated horses and were strongly correlated at the acute stage of the disease at which time laminar MMP-9 gene expression was significantly (15-fold) elevated. In contrast, BWE-treatment did not significantly elevate MMP-2 gene or protein expression in the laminae. Interestingly, MMP-9 that was present in extracts of laminae from BWE-treated horses at both the prodromal and acute stages of the disease was mainly in the zymogen form, suggesting that the accumulation of the MMP did not contribute to pathology during these stages. However, elevated presence of the MMP-9 zymogen in the tissue would predispose it to catastrophic damage should conditions arise that cleave the regulatory propeptide domain.     

Urinary Tract Infection in Dogs with Thoracolumbar Intervertebral Disc Herniation and Urinary Bladder Dysfunction Managed by Manual Expression,Indwelling Catheterization or Intermittent Catheterization

Objective— To evaluate risk factors for lower urinary tract infection (UTI) in dogs with intervertebral disc disease (IVDD) that had manual expression (ME), indwelling catheterization (IDC) or intermittent catheterization (ITC) for urinary bladder management. Study Design— Randomized‐clinical trial. Animals— Dogs (n=62) treated with urinary bladder dysfunction requiring surgery for IVDD and control dogs (n=30) that had surgery for reasons other than IVDD. Methods— Treated dogs were randomly assigned to ME, IDC, or ITC. Urine was collected for culture and antimicrobial susceptibility testing before and after treatment. Incidence and risk factors for UTI were evaluated. Bacterial isolates and antimicrobial resistance patterns were described. Results— Mean (±SD) time to urination was significantly longer for IDC dogs (7.4±2.75 days) than ME dogs (4.2±2.63) and ITC dogs (4.9±3.12). Thirteen treated dogs (21%) and no control dogs developed UTI: 4/25 (16%) ME, 8/25 (32%) IDC, and 1/12 (8%) ITC. Enterobacter sp. was most frequently isolated (4/13; 31%). Duration of treatment was the only risk factor for UTI and each additional day of treatment increased the risk of UTI 1.5 times. Conclusion— For dogs with acute IVDD, the duration of required urinary bladder management establishes the risk of UTI, not the urinary bladder management technique. Clinical Relevance— Duration of treatment for urinary bladder dysfunction is a risk factor for UTI in dogs recovering from acute IVDD. Treatment for urinary bladder management should be limited where possible and no method of treatment is preferred. For dogs managed by IDC, voluntary urination might occur before clinically suspected.     

Marked MMP-2 transcriptional up-regulation in mononuclear leukocytes invading the subarachnoidal space in aseptic suppurative Steroid-Responsive Meningitis–Arteritis in dogs

Canine Steroid-Responsive Meningitis–Arteritis (SRMA) is a suitable animal model for studies on the development of neutrophilic pleocytosis in aseptic meningitis. Samples of dogs in the acute phase of SRMA (n = 16) were examined for gene expression of matrix metalloproteinases (MMP)-2 and -9 and tissue inhibitors of metalloproteinases (TIMP)-1 and -2. Results were compared to those of dogs under glucocorticosteroid treatment for SRMA (n = 16) and dogs with other inflammatory and neoplastic diseases of the central nervous system (CNS) (n = 19). Samples included mononuclear (PBMCs) and polymorphonuclear cells (PBPMNs) of peripheral blood and cerebrospinal fluid white blood cells (CSF WBCs). In the acute phase of SRMA CSF WBCs showed mRNA expression for MMP-2 and -9 and TIMP-1 and -2, highlighting a contribution of these cells to the overall content of MMPs and TIMPs in CSF. MMP-2 mRNA levels in CSF WBCs were significantly up-regulated in comparison to PBMC expression levels, suggesting that MMP-2 is relevant for PBMC invasion into the subarachnoidal space and that the expression is influenced by migratory activity through the blood–CSF-barrier.     

T-helper cells from naive to committed

An endotoxin-induced mastitis model was used to study permeability changes associated with increased milk matrix metalloproteinase (MMP) activity in early inflammation. One quarter of two cows was inoculated with endotoxin (Escherichia coli 055:B5). Blood, milk, and whey were collected before and repeatedly after inoculation for 48 h. The profile and amounts of gelatinolytic MMPs were determined by zymography; gelatinase A (72 kD MMP-2) and gelatinase B (92 kD MMP-9) were identified by Western immunoblotting. Bovine serum albumin (BSA) and trypsin inhibitor capacity (TIC) were used as markers of capillary permeability with parallel examination of neutrophil penetration from blood to milk. Five clinical E. coli mastitis milk samples and five milk samples from cows with healthy udders were analyzed to detect whether increased levels of gelatinolytic MMP-2 and MMP-9 have a role in naturally occurring mastitis with endotoxin involvement. Milk MMP levels increased 2h after the endotoxin challenge. Both MMP-2 and MMP-9 were involved in this early proteolytic event. These increased MMP levels are associated with increased capillary permeability, evidenced first by the penetration of small molecular weight proteins, such as BSA and TIC. Later, 6-12h post endotoxin inoculation, neutrophilic leucocytes also entered the site, as they require larger tissue damage in basal membrane and interstitial tissue structures than BSA and TIC to extravasate. In naturally occurring disease, increased MMP-2 and MMP-9 levels were detected in milk. Thus, gelatinases, especially MMP-2, are involved in the early degradation of the blood-milk barrier, which precedes the penetration of blood-derived cellular components into milk in endotoxin-induced mastitis. In the future, measuring MMP in milk/whey might be a useful tool for diagnosing early mastitis.     

Concentrations of 15F2t isoprostane in urine of dogs with intervertebral disk disease

OBJECTIVE: To measure 15F(2t) isoprostane concentrations in the urine of dogs undergoing ovariohysterectomy (OHE) and dogs undergoing surgery because of intervertebral disk disease (IVDD) and to assess relationships between urinary concentrations of 15F(2t) isoprostanes and neurologic score in dogs with IVDD. ANIMALS: 11 dogs undergoing OHE and 32 dogs with IVDD undergoing hemilaminectomy. PROCEDURES: Paired urine samples were obtained at induction of anesthesia and approximately 1 hour after OHE (controls) and were collected from dogs with IVDD at induction of anesthesia (28 samples) and approximately 1 hour after hemilaminectomy (31 samples); 26 paired urine samples were obtained from dogs with IVDD. Urinary isoprostane concentrations were measured by use of a commercial ELISA, and results were adjusted on the basis of urinary creatinine concentrations. Differences in the mean isoprostane-to-creatinine ratio were analyzed. Neurologic score was determined in dogs with IVDD by use of the modified Frankel scoring system. RESULTS: Urinary isoprostane-to-creatinine ratios were significantly higher in dogs with IVDD than in control dogs before and after surgery. There was no significant difference between values before and after surgery for either group. There was a significant correlation of neurologic score and urinary isoprostane-to-creatinine ratio because dogs that had higher neurologic scores (ie, less severely affected) generally had higher isoprostane-to-creatinine ratios. CONCLUSIONS AND CLINICAL RELEVANCE: Urinary isoprostane-to-creatinine ratios were higher in dogs with IVDD before and after surgery. Analysis of these data suggests that dogs with IVDD are in a state of oxidative stress and that preemptive treatment with antioxidants warrants further investigation.