Agarose gel electrophoresis of cerebrospinal fluid proteins of dogs after sample concentration using a membrane microconcentrator technique

BACKGROUND: Cerebrospinal fluid (CSF) is produced in the cerebral ventricles through ultrafiltration of plasma and active transport mechanisms. Evaluation of proteins in CSF may provide important information about the production of immunoglobulins within the central nervous system as well as possible disturbances in the blood-brain barrier. OBJECTIVE: The objective of this study was to measure the concentration and fractions of protein in CSF samples using a membrane microconcentrator technique followed by electrophoresis, and to compare the protein fractions obtained with those in serum. METHODS: CSF samples from 3 healthy dogs and 3 dogs with canine distemper virus infection were concentrated using a membrane microconcentrator having a 0.5 to 30,000 d nominal molecular weight limit (Ultrafree, Millipore, Billerica, MA, USA). Protein concentration was determined before and after concentration. Agarose gel electrophoresis was done on concentrated CSF samples, serum, and serial dilutions of one of the CSF samples. RESULTS: Electrophoretic bands were clearly identified in densitometer tracings in CSF samples with protein concentrations as low as 1.3 g/dL. The higher CSF protein concentration in dogs with distemper was mainly the result of increased albumin concentration. CONCLUSION: The microconcentrating method used in this study enables characterization of the main protein fractions in CSF by routine electrophoresis and may be useful for interpreting the underlying cause of changes in CSF protein concentrations.     

High resolution protein electrophoresis of 100 paired canine cerebrospinal fluid and serum

This study was performed to investigate the diagnostic relevance of cerebrospinal fluid (CSF) high resolution electrophoresis. The laboratory technique was applied to 100 paired samples of canine CSF and serum, with paired samples tested during the same analytical run, as recommended in human medicine. Ninety four of the dogs had a neurological disease and 6 healthy dogs served as a control group. A strong linear correlation between CSF total protein concentration and the albumin quota (AQ) was found in the control group and in the inflammatory (infectious or noninfectious), neoplastic, and miscellaneous groups: AQ = 0.015 CSF total protein--0.102, r = 0.990. This correlation suggests that an increased CSF total protein concentration can be an indicator of blood brain barrier dysfunction. The highest median AQ value was found in the aseptic suppurative meningitis group, but no statistical differences were found between this and the other groups. The AQ, calculated with this technique, did not provide any additional information. Moreover, although unexpected, the electrophoretic profiles were not characteristic of any particular disease. In conclusion, this study did not confirm high resolution electrophoresis of paired CSF and serum samples to be a valuable ancillary diagnostic tool for canine neurological diseases.     

Cerebrospinal fluid analysis.

Accurate interpretation of cerebrospinal fluid (CSF) changes can only be made in the context of the differential diagnosis for each case. The routine analysis of CSF cell number and type as well as CSF total protein can provide information that suggests a specific mechanism or disease, but is often inconclusive. Further information obtained from CSF protein electrophoresis and immunoglobulin determination and calculation of an albumin quota and IgG index can lend additional support for the suspected mechanism of disease. Paired serum and CSF antibody titers for specific organisms can be useful to confirm the presence of a systemic or nervous system infection. Current research on detecting antibodies against nervous tissue components in CSF should result in better diagnostic capabilities and understanding of the pathophysiology of certain disorders in the future.     

Blood–brain‐barrier disruption in chronic canine hypothyroidism

Background: Central nervous system (CNS) manifestations of hypothyroidism have been associated with cerebrovascular complications. Reports of cerebrospinal fluid (CSF) abnormalities are rare in hypothyroid dogs. Objective: The aim of this study was to determine if chronic hypothyroidism causes blood–brain‐barrier (BBB) abnormalities that are detectable using indirect CSF biomarkers. Methods: The study included 18 normal, euthyroid, female mixed‐breed dogs. Hypothyroidism was induced by ¹³¹iodine administration in 9 dogs; 9 served as untreated controls. Evaluations included physical and neurologic examination, complete CSF analysis, serum and CSF protein electrophoresis, measurement of plasma vascular endothelial growth factor (VEGF) and serum S‐100B concentrations, and calculation of CSF albumin quota (AQ) and were conducted at baseline and 6, 12, and 18 months after induction of hypothyroidism. Data were analyzed using repeated measures ANOVA. Results: At baseline, differences between groups were not detected for any variable. Throughout the study, controls dogs remained free of neurologic disease and had test variables that remained within reference intervals. Two hypothyroid dogs developed CNS signs during the study, and evidence of cerebrovascular disease was found at necropsy. At 12 and 18 months, the CSF total protein, VEGF, S‐100B, and fractional albumin concentrations, and AQ were significantly higher (P<.04) in hypothyroid dogs than controls. Among test variables assayed in serum or plasma, the only significant difference was a higher S‐100B concentration in hypothyroid dogs (P=.003) at 18 months. Conclusions: BBB integrity is disrupted in chronic hypothyroidism. Significant increases in CSF concentrations of VEGF and S100‐B in hypothyroid dogs indicate dysfunction in both endothelial and glial elements of the BBB.     

Assessment of the IgG index in dogs by indirect immunoenzimatic assays as diagnostic tool for inflammatory diseases of central nervous system

The IgG index measures the intrathecal immunoglobulin production and it is a useful tool for diagnosis of inflammatory diseases involving the central nervous system. This index is based on the precise quantification of albumin and IgG in canine cerebrospinal fluid and serum. Here, we report the development of an indirect competitive ELISAs for the detection of both antigens. Thirty-two dogs were included in this study, divided into three experimental groups. Group A was composed of 22 healthy animals, as determined by standard clinical examination. In group B, six animals, presented neurological pathologies associated with endogenous IgG production and, in group C four animals presented neurological diseases or symptoms not associated with intrathecal IgG production. Cerebrospinal fluid and serum samples were obtained from these animals. As expected, by using the indirect ELISAs proposed here, the IgG indexes obtained in healthy animals (A) were 0.371+/-0.252 (SD). In B and C, the values (3.002+/-1.897; 0.36+/-0.306, respectively), were in agreement with the pathologic conditions of the individuals in each group. Thus, the immunometric competition ELISA methods proposed here allow the discrimination of abnormal intrathecal IgG production, in a variety of inflammatory pathologic conditions of the central nervous system.     

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.     

Cerebrospinal fluid cytology in canine neurologic disease

Samples of cerebrospinal fluid (CSF) of 93 dogs with neurologic diseases were examined by cytomorphologic technique, and the changes in the CSF were correlated with histopathologic examinations of the central nervous system (CNS). It was concluded that CSF examination is a significant aid in obtaining a neurologic diagnosis and that good correlation exists between the CSF changes and the pathologic changes in the CNS. The CSF examination allows making a diagnosis of encephalitis and differentiation between viral and other causes (although in mycotic infection the cell membrane preparation can be used to identify the cause directly), could allow making differentiation between congenital malformations and congenital degenerative disease, and helps in identifying physical spinal cord damage, differentiating it from muscular, neurogenic, or functional disorders clinically presented as spinal ataxia. The CSF cytologic examination can indicate the presence of hemorrhage in the CNS. There is not enough experience available in the diagnosis of brain tumors by means of CSF examination; however, in dogs with lymphosarcoma in the CNS, CSF cytologic changes can be diagnostic.     

Use of albumin quotient and IgG index to differentiate blood- vs brain-derived proteins in the cerebrospinal fluid of cats with feline infectious peritonitis

BACKGROUND: Inflammation of the central nervous system (CNS) is a frequent condition in cats but etiology often remains unsolved. Routine cerebrospinal fluid (CSF) analysis can be extended through the calculation of the albumin quotient (Q(alb)), a marker of the integrity of the blood-brain barrier (BBB), and IgG index, an estimate of intrathecal IgG synthesis. OBJECTIVES: The purpose of this study was to validate nephelometric methods for CSF protein analysis, and to use the Q(alb) and IgG index to discriminate blood- and brain-derived immunoglobulin fractions in cats with feline infectious peritonitis (FIP). METHODS: Cats presented to our clinic between 2001 and 2005 were included in the study based on clinical and laboratory data and histopathologic findings at necropsy. Cats were grouped as having nonneurologic disease (controls; n=37), brain tumors (n=8), FIP involving the CNS (n=12), and extraneural FIP (n=12). CSF-total protein (TP) was measured and albumin and IgG concentrations were measured in paired CSF/serum samples; Q(alb) and IgG index were calculated. Intraassay and interassay precision of the nephelometric assays were determined using pooled samples. RESULTS: Coefficients of variation for the nephelometric assays ranged from 2.7% to 7.2%. In control cats, CSF-TP concentration ranged from 0.06 to 0.36 g/L, Q(alb) ranged from 0.6 to 5.7 x 10(-3), and IgG index ranged from 0.3 to 0.6. Q(alb) and IgG index were significantly higher in cats with brain tumors and cats with CNS-FIP compared with other groups. Compared with control cats, pleocytosis was evident in 8 of 12 (67%) cats and CSF-TP was increased in 3 of 12 (25%) cats with CNS-FIP. CONCLUSION: Nephelometry is a reliable method for measurement of CSF protein, albumin, and IgG in cats. The Q(alb) and IgG index did not identify a CSF protein pattern specific for BBB dysfunction or intrathecal IgG synthesis in cats with CNS-FIP.     

Transient leakage across the blood-cerebrospinal fluid barrier after intrathecal metrizamide administration to dogs

Cerebrospinal fluid samples from 9 dogs given 84 mg of metrizamide/kg of body weight intrathecally were collected at intervals from 3 hours to 30 days after treatment and were compared with CSF samples collected before metrizamide treatment (base line) and with samples taken at similar intervals from 2 nontreated control dogs. Increased CSF albumin (mean 19.2 mg/dl) and immunoglobulin (Ig) G (mean 5.91 mg/dl) concentrations occurred 1 day after myelography, compared with base-line concentrations (6.15 and 1.24 mg/dl, respectively) and with concentrations from controls. Immunoglobulin M and IgA concentrations also were increased in some of these samples. However, immediately after myelography (3 hours after treatment) CSF albumin and IgG concentrations were comparable with those of controls, and these values returned to base line within 5 days of myelography and remained so for 30 days. A high correlation between albumin and IgG concentrations of individual CSF samples indicated the likelihood that leakage across the blood-CSF barrier was the origin of the increased values. A transient increase in CSF leukocytes, consisting of mononuclear cells and neutrophils, was also noticed 1 day after treatment but not at other times and not in controls. Nonsuppurative, predominately histiocytic meningitis of decreasing intensity was noticed in dogs euthanatized at 1 or 5 days, but not in dogs euthanatized at 10, 20, or 30 days after treatment. The meningeal cells stained intensely with periodic acid-Schiff stain and intracellular contrast medium was ultrastructurally apparent in phagolysosomes. Control dogs killed after 30 days did not have these changes. The intrathecal administration of metrizamide resulted in transient leakage of serum components into CSF and an accompanying transient meningitis.     

Immunoglobulin M-capture enzyme-linked immunosorbent assay testing of cerebrospinal fluid and serum from horses exposed to west nile virus by vaccination or natural infection

The West Nile (WN) virus, present in the United States since 1999, is a cause of encephalomyelitis in birds, alligators, humans, and horses. No data exist regarding detection of anti-WN virus immunoglobins in equine cerebrospinal fluid (CSF). The aims of this study were to evaluate the blood-brain barrier (BBB) in WN virus-infected (WNE) horses, to compare diagnostic testing in serum and CSF, and to describe the immunoglobulin M (IgM) response in serum and CSF of vaccinated horses. CSF was collected from the lumbosacral (LS) space (n = 13) or the allanto-occipital (AO) space (n = 14) of WNE horses. The albumin quotient (AQ) and IgG index were calculated, and the IgM-capture-enzyme-linked immunosorbent assay (MAC-ELISA) was used to detect anti-WN virus IgM in serum and CSF. CSF collected from the LS site had a higher (P < .02) IgG index compared to the AO site (0.34 +/- 0.04 versus 0.22 +/- 0.04 [mean +/- SE], respectively). The mean AQ, irrespective of collection site, did not exceed reference values. There was 100% agreement between CSF and serum testing for IgM by MAC-ELISA testing. However, the positive to negative antigen ratios were higher (P < .001) in CSF (34.5) versus serum (8.5), indicating lower nonspecific reactivity in CSF samples. Horses vaccinated against WN virus did not develop an IgM response at 1:400 mg/dL in serum; however, a few horses developed a weak IgM response in serum but not in CSF. In conclusion, MAC-ELISA testing of serum and CSF were equivocal. Also, examination of CSF data from WNE horses suggests a normal BBB integrity and increased intrathecal production of antibodies.     

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.     

Detection of oligoclonal bands in cerebrospinal fluid from German Shepherd dogs with degenerative myelopathy by isoelectric focusing and immunofixation

BACKGROUND: Detection of intrathecal IgG synthesis is important in evaluating inflammatory diseases in the central nervous system. Isoelectric focusing (IEF) is currently the most sensitive method to demonstrate intrathecal IgG synthesis and may have diagnostic value for German Shepherd degenerative myelopathy (GSDM). OBJECTIVE: The objective of this study was to adapt a modified IEF and immunofixation method for the detection of oligoclonal bands in cerebrospinal fluid (CSF) from dogs with GSDM. METHODS: Serum and lumbar CSF samples were collected from 6 German Shepherd dogs clinically diagnosed with GSDM. Samples were also collected from 6 clinically healthy dogs for comparison. The concentration of IgG was measured by quantitative ELISA and the concentration of total protein was measured by the Bradford protein assay. The presence of oligoclonal bands was evaluated by use of modified IEF followed by immunofixation. RESULTS: The concentrations of total protein and IgG, and the IgG/total protein ratio in CSF samples, were not significantly different between GSDM patients and control dogs. Four GSDM patients had oligoclonal bands in their CSF based on IEF-immunofixation. No oligoclonal bands were found in CSF from control dogs. CONCLUSION: The results suggest that the detection of oligoclonal bands by IEF-immunofixation may have diagnostic value for GSDM, and support the idea that humoral immune responses may play a role in the pathogenesis of GSDM.     

Cerebrospinal fluid glutamine,tryptophan, and tryptophan metabolite concentrations in dogs with portosystemic shunts

OBJECTIVE: To determine whether glutamine (GLN), tryptophan (TRP), and tryptophan metabolite concentrations are higher in cerebralspinal fluid (CSF) dogs with naturally occurring portosystemic shunts (PSS), compared with control dogs. ANIMALS: 11 dogs with confirmed PSS and 12 control dogs fed low- and high-protein diets. PROCEDURE: Cerebrospinal fluid and blood samples were collected from all dogs. Serum and CSF concentrations of GLN, alanine, serine, TRP, 5-hydroxyindoleacetic acid (5-HIAA), and quinolinic acid (QUIN) were measured. RESULTS: Cerebrospinal fluid concentrations of GLN, TRP, and 5-HIAA were significantly higher in PSS dogs, compared with control dogs fed high- or low-protein diets. Cerebrospinal fluid QUIN concentration was significantly higher in PSS dogs, compared with control dogs fed the low-protein diet. Serum QUIN concentration was significantly lower in PSS dogs, compared with control dogs fed either high- or low-protein diets. CONCLUSIONS AND CLINICAL RELEVANCE: An increase in CNS GLN concentration is associated with high CSF concentrations of TRP and TRP metabolites in dogs with PSS. High CSF 5-HIAA concentrations indicate an increased flux of TRP through the CNS serotonin metabolic pathway, whereas high CSF QUIN concentrations indicate an increased metabolism of TRP through the indolamine-2,3-dioxygenase pathway. The high CSF QUIN concentrations in the face of low serum QUIN concentrations in dogs with PSS indicates that QUIN production from TRP is occurring in the CNS. High concentrations of QUIN and other TRP metabolites in the CNS may contribute to neurologic abnormalities found in dogs with PSS and hepatic encephalopathy.     

Determination of immunoglobulin A concentrations in the serum and cerebrospinal fluid of dogs: an estimation of its diagnostic value in canine steroid-responsive meningitis-arteritis

Previous studies on canine steroid-responsive meningitis-arteritis (SRMA) suggested that elevation of immunoglobulin A (IgA) concentrations in both serum and cerebrospinal fluid (CSF) is specific for SRMA throughout the different disease stages. Recent studies however have raised concerns about the value of this test. The purpose of this study was to investigate the diagnostic value of IgA concentration testing in paired CSF and serum samples. IgA concentrations of 525 paired canine CSF and serum samples were evaluated. Samples were obtained from dogs with SRMA (n=311) and dogs with miscellaneous conditions (n=214) such as other central nervous system (CNS) inflammatory diseases (n=34), CNS tumours (n=46), idiopathic epilepsy (n=42), intervertebral disc disease (n=46) and non-CNS diseases (n=46). Serum IgA concentrations were significantly higher in dogs with untreated SRMA compared to those with other diseases. IgA CSF concentrations were significantly higher in dogs with SRMA compared to other disease categories, with the exception of inflammatory CNS disease. The sensitivity for IgA concentrations in serum and CSF was 91% with a specificity of 78%. Analysis of 525 paired samples confirmed that IgA concentrations were higher in dogs with SRMA. Calculation of the diagnostic value of IgA concentration confirmed that the test is highly sensitive for SRMA. Testing paired CSF and serum samples for IgA is still recommended for the diagnosis of suspected cases of SRMA.     

Serum proteins in healthy Basenjis and Basenjis with chronic diarrhea

A serologic survey was conducted to characterize the electrophoretic patterns of serum proteins in healthy Basenji dogs (n = 137) and Basenjis with chronic diarrhea (n = 32). Serum protein electrophoresis values for Basenjis were similar to previously reported values from dogs of other breeds. Dogs with histologically confirmed lymphocytic-plasmacytic enteritis had a statistically significant (P less than 0.05) decrease in total protein, albumin, and albumin/globulin ratio. alpha 2-Globulin and gamma-globulin values were significantly increased in old dogs (9 years of age or older) with lymphocytic-plasmacytic enteritis. Although not statistically significant, gamma-globulin values were generally increased in Basenjis with lymphocytic-plasmacytic enteritis when compared with the values in age-matched clinically healthy Basenjis.     

Autoantibodies against structures of the central nervous system in steroid responsive meningitis-arteriitis in dogs

Steroid-responsive meningitis-arteriitis (SRMA) is a disease of dogs familiar in small animal practice for decades. A combined evaluation of IgA in serum and cerebrospinal fluid (CSF) is an important diagnostic tool. It is suspected that immunpathological mechanisms are involved in the pathogenesis of SRMA because of the marked response to steroids. Excessive production of IgA seems to play a central role and might be caused by an immune reaction to self-antigens of the central nervous system (CNS). To test this hypothesis, we analyzed CSF samples from 55 dogs with SRMA using the western blot method. After blotting canine brain tissue, IgA, IgM and IgG of the CSF samples were tested for their binding to CNS antigens. We also evaluated CSF samples from 45 dogs with other brain diseases, including different encephalitides and intracranial tumors, and from healthy dogs as controls. Positive reactions (mostly IgA) were observed in the CSF samples from dogs with SRMA, different encephalitides and brain tumors (a total of 8% positive samples). The occurrence of autoantibodies against CNS structures was significantly higher in the control group "other brain diseases" than in the SRMA group (p = 0.0135). There was no significant difference in the number of positive samples between dogs with SRMA and the negative control group (healthy dogs, p = 0.1535). Despite the small number of positive samples, only dogs with abnormal findings in the CSF analysis also had autoantibodies in the CSF. There was no significant correlation between the occurrence of autoantibodies and levels of IgA, protein content and cell counts in the cerebrospinal fluid. However, there was a certain trend toward positive reactions in CSF samples with high protein content. The occurrence of autoantibodies in dogs with SRMA thus seems to be an epiphenomenona rather than the cause of the disease.     

Canine serum protein patterns using high-resolution electrophoresis (HRE)

Serum protein values were determined in 26 healthy dogs using agarose gel electrophoresis (SPE), splitting the electrophoretic separation into six regions: albumin, alpha(1), alpha(2), beta(1), beta(2)and gamma globulins. High-resolution electrophoresis (HRE) was used to separate single proteins. Serum proteins from dogs (26 healthy and 20 affected by various diseases) were then characterized by electrophoretic immunofixation (IFE) and Sudan black staining on HRE film. Haemoglobin and normal canine plasma and serum were used to identify haptoglobin and fibrinogen, respectively.In the standard pattern, determined by HRE, the following proteins were identified: albumin, alpha(1)-lipoprotein (alpha(1)-region), haptoglobin and alpha(2)-macroglobulin (alpha(2)-region), beta -lipoprotein and C3 (beta(1)-region), transferrin and IgM (beta(2)-region), IgG (mostly in gamma -region and partly in beta(2)-region). The HRE pattern shown by healthy dogs could be compared with those of dogs affected by various diseases to obtain clinical information.     

Postprandial changes in leptin concentrations of cerebrospinal fluid in dogs during development of obesity

OBJECTIVE: To evaluate postprandial changes in the leptin concentration of CSF in dogs during development of obesity. ANIMALS: 4 male Beagles. PROCEDURES: Weight gain was induced and assessments were made when the dogs were in thin, optimal, and obese body conditions (BCs). The fat area at the level of the L3 vertebra was measured via computed tomography to assess the degree of obesity. Dogs were evaluated in fed and unfed states. Dogs in the fed state received food at 9 AM. Blood and CSF samples were collected at 8 AM, 4 PM, and 10 PM. RESULTS: Baseline CSF leptin concentrations in the thin, optimal, and obese dogs were 24.3 +/- 2.7 pg/mL, 86.1 +/- 14.7 pg/mL, and 116.2 +/- 47.3 pg/mL, respectively. In the thin BC, CSF leptin concentration transiently increased at 4 PM. In the optimal BC, baseline CSF leptin concentration was maintained until 10 PM. In the obese BC, CSF leptin concentration increased from baseline value at 4 PM and 10 PM. Correlation between CSF leptin concentration and fat area was good at all time points. There was a significant negative correlation between the CSF leptin concentration-to-serum leptin concentration ratio and fat area at 4 PM; this correlation was not significant at 8 AM and 10 PM. CONCLUSIONS AND CLINICAL RELEVANCE: Decreased transport of leptin at the blood-brain barrier may be 1 mechanism of leptin resistance in dogs. However, leptin resistance at the blood-brain barrier may not be important in development of obesity in dogs.     

The qualitative analysis of canine immunoglobulins and myeloma proteins by immunofixation

The technique of immunofixation was used to locate the immunoglobulin species in canine sera following agarose electrophoresis. The myeloma proteins in two dogs were identified as immunoglobulin G, and a polygonal gammopathy was characterized by this method.