Cerebrospinal Fluid

2. Blood-Csf Barrier

The epithelial cells of the circumventricular organs form one part of the blood-CSF interface. The circumventricular organs, which include the four choroid plexuses, the median eminence, the neural lobe of the hypophysis, and other specialized areas, border the brain ventricles and are involved with specific secretory activities that appear to require a direct contact with plasma. The capillaries within these organs are fenestrated, similar to capillaries in other organs of the body. Overlying each of the organs are specialized epithelial cells joined by intercellular tight junctions at their apical (ventricular) borders. These epithelial cells also are characterized by an abundance of intracellular organelles and lysosomes. These organelles are probably an important aspect of the barrier and secretory functions of these cells (Davson and Segal, 1996; Fishman, 1992; Milhorat, 1987; Rosenberg, 1990). The choroid plexuses are the major source of CSF. They are formed by evaginations of the ependyma and the pial blood vessels into the ventricles, and they consist of a single row of cuboidal, specialized epithelial cells thrown into villi around a core of blood vessels and connective tissue. The apical (ventricular) surface of the epithelial cells has a brush border of microvilli with occasional cilia. The basal and lateral cell surfaces have numerous infoldings. Overall, the structure of these cells resembles other epithelia specialized for fluid transport, such as proximal renal tubular epithelium (Davson and Segal, 1996; Fishman, 1992; Milhorat, 1987; Rosenberg, 1990). Autonomic nerve terminals have also been identified in the choroid plexus, but their function is unclear (Fishman, 1992; Nilsson et al., 1992).

The second part of the blood-CSF interface is formed by the arachnoid membrane at the arachnoid villi. These villi are microscopic evaginations of the arachnoid membrane into the lumen of the dural sinuses. The barrier function of these arachnoid cells is demonstrated by their tight junctions. Their transport function is indicated by giant intracellular vacuoles, some of which have both basal and apical openings, and pinocytotic vesicles. The sinus surface of a villus is covered by sinus endothelium (Milhorat, 1987; Rosenberg, 1990). Endothelium-lined channels may link directly with the subarachnoid space (Bell, 1995; Davson and Segal, 1996). Arachnoid villi are not limited to intracranial venous sinuses but also are present at the spinal nerve roots penetrating into the spinal veins (Bell, 1995; Milhorat, 1987).

1. Albumin

With electrophoretic techniques, protein in the CSF can be separated into prealbumin, albumin, and alpha, beta, and gamma globulins. The major protein in CSF is albumin, which is synthesized only in the liver. The limited entry of albumin into the CSF is dependent on the blood-brain/CSF barrier to macromolecules. When total CSF protein increases, the albumin concentration increases disproportionately. This phenomenon illustrates the role of molecular size in determining the distribution of serum proteins into the CSF (Felgenhauer, 1974).

2. Alpha and Beta Globulins

Immunoelectrophoresis can separate the alpha and beta globulins into several proteins (Table 26.9). The origin of tau protein (beta₂ transferrin) is uncertain. This protein may be modified serum transferrin (beta₁ transferrin) or it may be a unique protein, “tau protein,” in the CSF (Fishman, 1992). In veterinary and human medicine, no correlation has been made between changes in the concentrations of these globulins and specific neurological disease (Fishman, 1992; Sorjonen et al., 1991). Thus, their measurement has limited clinical use at this time.

3. Gamma Globulins

Because of the changes found in association with multiple sclerosis and other inflammatory diseases, the gamma globulins have received a great deal of attention. Electrophoretic techniques define the gamma globulins as a heterogeneous group of proteins with migrations at similar rates (see Table 26.9). The gamma globulin fraction contains immunoglobulins. Immunological assays identify three major immunoglobulins in normal CSF: IgG, IgM, and IgA. Minute amounts of other immunoglobulins have also been detected in normal CSF (Fishman, 1992; Kjeldsberg and Knight, 1993).

2. Total leukocyte and erythrocyte counts

Electronic cell or particle counters are typically not sensitive enough to be used for enumeration of cells in CSF. The level of background counts with these counters is frequently in excess of the counts present in the majority of CSF samples that are analyzed. Therefore, cells are usually counted with a standard hemacytometer chamber with Neubauer ruling (Brobst, 1989; Cook and DeNicola, 1988; Jamison and Lumsden, 1988). The chamber is charged with undiluted fluid. Ideally, the cells are allowed to settle for 10 min in a humidified environment. This allows all the cells to be visible in the same plane of focus. The cells in the nine largest squares on both sides of the chamber are counted (18 squares in total) and the result multiplied by 0.55 to obtain the number of cells per microliter. Alternatively, the cells in nine large squares are counted and the number multiplied by 1.1 to determine the count per microliter. To the untrained observer, unstained leukocytes and erythrocytes may be difficult to differentiate. Leukocytes are larger, and the presence of nuclei gives them a more granular appearance than erythrocytes. With experience, nuclear morphology can often be appreciated (Cook and DeNicola, 1988). The cytoplasmic border is usually slightly irregular. In contrast, erythrocytes are usually smaller, smooth, and refractile, although they may become crenated upon standing (Jamison and Lumsden, 1988). Differentiating nucleated cells and erythrocytes in a hemacytometer chamber can be expedited by staining with New Methylene Blue before counting (Fry et al., 2006). This latter technique can be used without significant dilutional effects.

A laser based cell counter and dedicated software are used to count and differentiate cells in human CSF (Aune et al., 2004; Mahieu et al., 2004). This technique has the advantage of markedly superior precision and accuracy. The same methodology has been used to assess canine CSF (Ruotsalo et al., 2005). Although there was good correlation between the leukocyte and erythrocyte concentrations when compared with standard hemacytometer methods, the current software algorithms were not suitable for determining an accurate differential count in canine CSF. Additionally, it is likely that the cost and logistics of this methodology will preclude routine use in veterinary medicine.

3. Cytological examination

There are a variety of methods to facilitate cytological examination of CSF (Barrett and King, 1976; Ducos et al., 1979; Grevel, 1991; Hansen et al., 1974; Jamison and Lumsden, 1988; Kolmel, 1977; Roszel, 1972; Sornas, 1967; Steinberg and Vandevelde, 1974; Woodruff, 1973). There is controversy as to which method is optimal, and all have their strengths and weaknesses. Methods include simple centrifugation, sedimentation, and variations thereof; membrane filtration; and cytocentrifugation (Jamison and Lumsden, 1988). Consult these references for specific methodological details. Simple centrifugation usually produces slides that are unsatisfactory for cytological examination. The chief advantage of membrane filtration techniques is excellent cellular recovery with yields approaching 90% to 100% (Barrett and King, 1976). However, the methodology is laborious and time consuming, the cellular morphology relatively poor, many cells are partly hidden in the filter substance, which itself stains variably, and the technique requires specialized, nonroutine staining techniques that most veterinary clinical pathologists do not have experience or expertise in interpreting. For these reasons, they are not recommended. Cytocentrifugation (Hansen et al., 1974; Woodruff, 1973) is the method of choice in both human (Fishman, 1992; Kjeldsberg and Knight, 1993) and veterinary medicine (Christopher et al., 1988; Jamison and Lumsden, 1988). It is rapid, simple, and produces slides with good cytological detail. The technique is enhanced by the addition of protein to the CSF sample before centrifugation, which helps to preserve cell morphology. Conditions of cytocentrifugation vary from laboratory to laboratory. We prefer the method described by Rand and colleagues (Rand et al., 1990b). The disadvantages of cytocentrifugation are the expense of the instrument and the relatively low cell yield. In one comparative study (Barrett and King, 1976), the following cell yields were determined: millipore filtration 81±3% (SEM), nucleopore filtration 69±3%, and cytocentrifugation 11±1%. The Sornas method of centrifugation results in a cell yield, after staining, of approximately 20% (Sornas, 1967). The sedimentation technique of Sayk, modified by Kölmel (Grevel, 1991; Grevel and Machus, 1990; Kolmel, 1977), results in a yield of approximately 30% (Kolmel, 1977), although this can be increased to almost 90% if a membrane filter is substituted for direct sedimentation onto a slide. We have some experience with the Kölmel apparatus and technique and have found the cell morphology to be at least as good as cytocentrifugation with an apparently superior cell yield, although this needs to be confirmed with controlled comparative studies. Therefore, most studies suggest that sedimentation techniques result in greater cell yields than does cytocentrifugation. However, there is at least one study that found the yield of cytocentrifugation to be marginally higher than sedimentation (Ducos et al., 1979). Standard Romanowsky stains are recommended for staining of slides. They provide good cellular detail on air-dried CSF preparations and are familiar to most observers. These stains include the Wright's and Wright–Giemsa staining methods as well as a variety of rapid staining methods including Diff-quik and Camco-quik (Jamison and Lumsden, 1988).

4. Immunocytochemistry

In people, the value of cytological diagnosis of CSF can be improved if morphological studies are appropriately supplemented by immunocytochemistry (Kjeldsberg and Knight, 1993). Immunophenotypic studies of cytocentrifuge slides are useful in the differential diagnosis of leukemia, lymphosarcoma, primary brain tumors, and metastatic tumors (Bigner, 1992; Bigner and Jonston, 1981; Jorda et al., 1998; Kjeldsberg and Knight, 1993; Tosaka et al., 2001). Few veterinary studies document CSF immunocytochemistry. In dogs, immunophenotyping is useful in the diagnosis of lymphoma and infiltrative leukemia² and histiocytic sarcoma, both postmortem (Zimmerman et al., 2006) and antemortem.³

Panels of monoclonal antibodies are typically used for the immunocytochemical assessment of CSF. The greatest limitation is therefore the volume and cellularity of the specimen available for the marker studies. Undifferentiated tumor panels frequently include leukocyte common antigen and cytokeratin antibodies. These can be helpful in distinguishing single carcinoma cells from lymphocytes or monocytes (Bigner, 1992; Bigner and Jonston, 1981; Kjeldsberg and Knight, 1993). Glial fibrillary acidic protein has proven to be helpful in distinguishing a glial origin, but there are currently no specific markers to distinguish primary brain tumors. Immunocytochemistry can be used also to characterize the lymphocyte subpopulations present in CSF. Seventy-five to 95% of the lymphocytes found in normal human CSF are T cells, with a mean of approximately 85% (Kjeldsberg and Knight, 1993). Within the population of T cells, T-helper cells predominate and account for up to 88% of T cells. Alterations of these percentages have been shown to have significant associations with disease in people (Kjeldsberg and Knight, 1993). Similar studies assessing CSF lymphocyte subset alterations in disease are lacking in domestic animals but may be useful. Lymphocyte subset distribution has been assessed in the brains of dogs with different types of diseases (Tipold et al., 1999). T cells predominated in viral encephalitides, whereas B cells predominated in bacterial and protozoal diseases and in steroid responsive meningitis-arteritis (Tipold et al., 1999). However, it has not been determined if similar changes are reflected in the CSF. Lymphocyte subset distribution has been assessed in normal dogs and horses (Duque et al., 2002; Furr et al., 2001; Tipold et al., 1998) Similar to people, T lymphocytes predominate in canine CSF but are present as a lower percentage than in people, accounting for approximately 50% to 60% of lymphocytes (Duque et al., 2002; Tipold et al., 1998). However, there appears to be much individual variation that may complicate use of this type of assessment clinically (Tipold et al., 1998). Horses appear to be more similar to people, with T cells accounting for approximately 80% of lymphocytes in the CSF (Furr et al., 2001). However, in contrast to people, CD8+T cells in normal equine CSF may constitute a greater subset of T cells, accounting for approximately 30% of CSF T cells in one study (Furr et al., 2001). In people, most central nervous system lymphomas are B cell in origin; immunocytochemistry assessing immunoglobulin light chain expression can be used to document monoclonality (Bigner, 1992; Bigner and Jonston, 1981). This is strong evidence (but not definitive proof) of malignancy. This assessment cannot be made in most domestic animals because of the marked light chain skewing that exists normally in these species (Arun et al., 1996: Butler, 1998, #387). The B cell origin of the lymphocytes can also be confirmed with demonstration of immunoglobulin light chain expression. For patients with T cell lymphomas, marker studies can be more difficult to interpret as T cells predominate in normal and inflammatory CSF (Kjeldsberg and Knight, 1993). If there is systemic involvement, then comparison with the peripheral phenotype is useful to confirm presence in the CSF. Immunocytochemistry has also been utilized to detect infectious agents such as cytomegalovirus and mycobacterium tuberculosis in human patients (Stark et al., 1993; Sumi et al., 2002) and distemper virus in dogs (Abate et al., 1998).

3. Albumin and the csf/Serum Albumin Index

Because albumin is synthesized only extrathecally, increased CSF albumin indicates damage to the blood-brain/CSF barriers, intrathecal hemorrhage, or a traumatic CSF tap. In these conditions, albumin will leak into the CSF in general proportion to its serum concentration. Therefore, in the absence of intrathecal hemorrhage (pathological or iatrogenic), the ratio of CSF albumin to serum albumin can be used as an indicator of barrier dysfunction (Link and Tibbling, 1977; Tibbling et al., 1977). This ratio is also called the albumin index (a.k.a. albumin quota, albumin quotient) and is calculated as follows (Kjeldsberg and Knight, 1993):

Values above the normal range indicate increased barrier permeability. The use of this index is potentially limited, however, because the large variability of CSF albumin in normal animals (at least in dogs and horses) (Andrews, Geiser, Sommardahl, Green, & Provenza (1994), Andrews, Maddux, & Faulk (1990a); Bichsel et al., 1984b; Krakowka et al., 1981) results in a large variability in the values for this index (Davson and Segal, 1996). In people, the albumin index is age dependent, being highest in newborns, and lowest in childhood. The albumin index increases with age (Deisenhammer et al., 2006).

4. Quantitative Measurement of Immunoglobulins

1. Neutrophilia

A marked pleocytosis with neutrophil predominance suggests either bacterial meningitis (Kjeldsberg and Knight, 1993; Kornegay et al., 1978) or suppurative, nonseptic (corticosteroid responsive) meningitis (Meric (1988), Meric (1992a); Tipold and Jaggy, 1994). Total leukocyte counts in excess of 2000 cells per microliter are frequently encountered in these diseases and may even exceed 10,000 cells per microliter (Meric, 1992a). Observation of bacteria or a positive culture confirms septic meningitis. In our experience, bacteria are more commonly observed in the CSF of large animals afflicted with septic meningitis than in dogs or cats with septic meningitis. Neutrophil nuclear morphology is often used as criteria for determining the likelihood of sepsis with nuclear degenerative changes or karyolysis interpreted as evidence of bacterial disease. However, the neutrophils in confirmed cases of septic meningitis in dogs and cats are frequently well preserved, especially if there has been prior therapy. Therefore, absence of bacteria or degenerative nuclear changes in neutrophils cannot be used to unequivocally exclude a diagnosis of septic meningitis, although it does make it less likely. In people, acute viral meningoencephalitis initially causes a neutrophilic pleocytosis (Converse et al., 1973; Fishman, 1992; Kjeldsberg and Knight, 1993) that may persist from a few hours to several days before the development of the more typical mononuclear reaction. A similar phenomenon is documented in animals (Green et al., 1993). Occasionally, distemper virus infection causes massive encephalomalacia (Vandevelde and Spano, 1977) resulting in a neutrophilic pleocytosis, in contrast to the more typical moderate mononuclear pleocytosis. Central nervous system neoplasia may result in a neutrophil predominance in the CSF, especially if there is significant necrosis and inflammation associated with the tumor. Moderate to marked pleocytosis with neutrophil predominance may be noted in dogs with meningioma (Bailey and Higgins, 1986b). However, in another study of dogs with meningioma, about 30% of dogs had a normal CSF analysis (Dickinson et al., 2006). In this study, a significant association between meningiomas in the caudal portion of the cranial fossa and an elevated CSF nucleated cell count was found; but only 19% of the dogs had an elevated total nucleated white cell count with a predominance of neutrophils (Dickinson et al., 2006).

Canine intervertebral disk disease is associated with variable alterations in CSF that depend on factors such as disease severity and chronicity (Thomson et al., 1989). Acute severe disease may result in counts greater than 50 cells per microliter with more than 50% neutrophils (Thomson et al., 1989). This finding may be a reflection of acute inflammation secondary to trauma that may be exacerbated by myelomalacia in some instances. The authors have seen a similar phenomenon associated with fibrocartilaginous embolic myelopathy in dogs. A neutrophilic pleocytosis of varying severity often occurs following myelography with iodinated contrast agents (Carakostas et al., 1983; Johnson et al., 1985; Widmer et al., 1992). These changes usually peak at 24 h postmyelogram (see Section VII for further details). Similarly, a neutrophilic pleocytosis has been observed postictally in people. We have occasionally observed similar findings in dogs (see Section VII).

2. Lymphocytosis

Alterations in both numbers and morphology of lymphocytes (see Section III.B) in the CSF occur in a variety of diseases. Central nervous system viral infections often result in a predominantly lymphocytic pleocytosis, documented in dogs (Vandevelde and Spano, 1977), cats (Dow et al., 1990; Rand et al., 1994b), horses (Green et al., 1992; Hamir et al., 1992; Wamsley et al., 2002), sheep, goats (Brewer, 1983), and numerous other species. In people, CSF lymphocytosis has been observed in bacterial meningitis following antibiotic therapy (Cargill, 1975; Converse et al., 1973; Fishman, 1992; Kjeldsberg and Knight, 1993), indicating that therapy and chronicity can alter the CSF findings. A similar finding has been reported in dogs (Sarfaty et al., 1986; Tipold and Jaggy, 1994) and calves (Green and Smith, 1992). We have noted that dogs with chronic or acute on chronic type I intervertebral disk disease have a pleocytosis that is more commonly lymphocytic than neutrophilic (Windsor et al., 2007). The CSF findings with granulomatous meningoencephalitis (GME) are somewhat variable, but a marked lymphocytic pleocytosis is common (Bailey and Higgins, 1986a; Thomas and Eger, 1989). Similarly, dogs with necrotizing meningoencephalitis (small breed dogs including pug dogs, Yorkshire terriers, and Maltese terriers) frequently have a marked lymphocytic pleocytosis (Cordy and Holliday, 1989; Stalis et al., 1995; Tipold et al., 1993a).

3. Eosinophilia

Eosinophils are not present in normal, uncontaminated (by blood) CSF. Single eosinophils are occasionally noted on cytocentrifuge slides from animals with normal CSF (normal nucleated counts and protein concentration). Although the presence of eosinophils in CSF is abnormal and evidence of underlying disease, no diagnostic specificity is associated with their presence in human CSF, as they can be found in a variety of diseases (Bosch and Oehmichen, 1978). Additionally, CSF eosinophilia and peripheral blood eosinophilia do not necessarily occur together and if they do, no positive correlation exists between the magnitude of peripheral blood eosinophilia and the severity of the CSF eosinophilia (Bosch and Oehmichen, 1978; Smith-Maxie et al., 1989). In one case series of eight dogs with eosinophilic meningoencephalitis, five of eight had concurrent peripheral eosinophilia, but no correlation was present between the peripheral and CSF eosinophil counts. The two dogs with the highest CSF eosinophil counts had peripheral eosinophil counts within normal reference limits. In people, central nervous system invasion by parasites, especially Angiostrongylus cantonensis, is the most frequent cause of eosinophilic pleocytosis; in many of these cases eosinophils predominate in the CSF differential cell count (Bosch and Oehmichen, 1978; Kuberski, 1979). A marked eosinophilic pleocytosis is also reported in dogs with neural angiostrongylosis (Lunn et al., 2003; Mason, 1989). CSF eosinophilia can also occur in association with bacterial, fungal, and viral infections and hence can be seen concurrently with suppurative, granulomatous, and lymphocytic inflammatory processes of the central nervous system (Jamison and Lumsden, 1988; Smith-Maxie et al., 1989). However, in many of these cases, eosinophils represent less than 5% of the total cell count in CSF (Bosch and Oehmichen, 1978; Smith-Maxie et al., 1989). Other documented causes in people include neurosyphilis, tuberculosis, rickettsial disease, foreign body reactions to shunt tubes, intrathecal penicillin or contrast agents, hypereosinophilic syndrome, multiple sclerosis, lymphoma, Hodgkin's disease, leukemia, melanoma, disseminated glioblastoma, idiopathic, and systemic allergic reactions (Bell et al., 2006; Fishman, 1992; Kjeldsberg and Knight, 1993; Kuberski, 1979; Smith-Maxie et al., 1989). In animals, CSF pleocytosis that consists predominantly, or almost exclusively, of eosinophils is rare. We have personally seen CSF eosinophilia with marked eosinophil predominance in association with idiopathic or steroid responsive eosinophilic meningoencephalitis (Smith-Maxie et al., 1989), canine neural angiostrongylosis (Mason, 1989), and histopathologically confirmed canine CNS neosporosis.⁴ Golden retriever dogs and rottweilers may be predisposed to idiopathic or steroid responsive eosinophilic meningoencephalitis (Bennett et al., 1997; Smith-Maxie et al., 1989). Pleocytosis with eosinophil predominance has also been described in central nervous system cryptococcosis (Vandevelde and Spano, 1977), although this finding is not common in our experience. Other documented causes of CSF eosinophilia (though not necessarily predominance) in animals include bacterial encephalitis, distemper, rabies, toxoplasmosis, neosporosis, cuterebral encephalitis, central nervous system nematodiasis and cestodiasis, protothecosis, granulomatous meningoencephalomyelitis, lymphoma, astrocytoma, cerebral infarction, canine neural angiostrongylosis, and salt poisoning (Chrisman, 1992; Darien et al., 1988; Jamison and Lumsden, 1988; Lester, 1992; Mac Donald et al., 1976; Mason, 1989; Oruc and Uslu, 2006; Smith, 1957; Tyler et al., 1980; Vandevelde and Spano, 1977).

3. Increased csf albumin and Albumin Index

Elevation of CSF albumin (which originates in the serum), and consequently an increased albumin index, indicates dysfunction of the blood-brain/spinal cord/CSF barriers or contamination of the CSF by blood (from intrathecal hemorrhage or traumatic spinal tap). Barrier damage occurs in most types of neurological disorders, including inflammatory diseases, neoplasia, trauma, compression, and occasionally metabolic diseases (Bichsel et al., 1984b; Krakowka et al., 1981; Sorjonen, 1987; Sorjonen et al., 1991).

4. Increased csf igG and igG index

The CSF IgG can be increased by movement of protein across damaged blood-brain/CSF barriers, intrathecal hemorrhage (pathological or iatrogenic), or intrathecal IgG synthesis. An elevated CSF IgG content and increased IgG index, indicating intrathecal IgG synthesis, are typical for infectious inflammatory diseases (Tipold, Pfister, & Vandevelde (1993b), Tipold, Pfister, Zurbriggen, & Vandevelde (1994)). In contrast, animals with noninflammatory diseases usually have normal IgG indices (Tipold et al., 1993b). In a few animals with noninfectious disorders mild intrathecal IgG synthesis occurs, reflecting the presence of inflammatory infiltrates around the lesion (Tipold et al., 1993b). Therefore, the IgG index is often useful for distinguishing between inflammatory and noninflammatory lesions, which is not always possible on the basis of CSF cell counts alone (Bichsel et al., 1984b). In one study (Tipold et al., 1993b), 7 of 66 dogs with inflammatory lesions had no pleocytosis but had an elevated IgG index; in contrast, 17 of 32 dogs with noninflammatory disease had pleocytotic CSF and a normal IgG index. The authors of this study consider an IgG index≥2.8 as proof of intrathecal synthesis allowing a diagnosis of meningoencephalomyelitis, and an IgG index between 1.3 and 2.8 as suggestive of inflammatory disease. In a few dogs with marked inflammatory lesions and intrathecal IgG production, the IgG index may not be elevated because of marked IgG exudation against which the local IgG synthesis is undetectable (Bichsel et al., 1984b). Traumatic puncture and red blood cell contamination of the CSF can artifactually increase the IgG index. Additionally, the normal IgG index of cerebellomedullary fluid and lumbar fluid are likely to be different because of the different protein concentrations of these fluids.

5. Classification of disease based on Albumin Index and IgG Index

Alterations of the albumin index and the IgG index can be grouped into three pathogenetical categories: (1) blood-brain/CSF barrier disturbance (increased albumin index), (2) intrathecal IgG synthesis (increased IgG index), and (3) barrier disturbance combined with intrathecal IgG production (both indices increased). These categories correlate somewhat with certain types of diseases. Barrier disturbance may be seen in degenerative, inflammatory, metabolic, space-occupying, vascular, and traumatic conditions (Bichsel et al., 1984b; Sorjonen, 1987; Sorjonen et al., 1991). Intrathecal IgG synthesis is typical of inflammatory conditions (Tipold et al., 1994) but also occurs in noninfectious disorders that have secondary inflammation such as some tumors (Tipold et al., 1993b). Barrier disturbance coupled with intrathecal IgG production is typical of infectious-inflammatory diseases (Bichsel et al., 1984b).

6. Increased csf igM, igA, and igM and igA indices

The immunoglobulins IgM and IgA may be increased in the CSF of animals with inflammatory neurological disease. A study of 69 dogs with inflammatory disease detected IgM elevations in 16 dogs and IgA elevations in 40 (Tipold et al., 1994). An increased CSF IgM index is considered by some investigators to be a good indication of recent or persistent immunological stimulation in people (Sharief and Thompson, 1989). In contrast, one study reported that IgM was present through all stages of human herpes and bacterial meningitis, and increased and decreased with IgG (Felgenhauer, 1982). Perhaps a transition from IgM to IgG production does not occur in the central nervous system (Tipold et al., 1994), or perhaps, in the presence of a normal or near-normal blood-CSF barrier, IgM accumulates in the CSF (Felgenhauer, 1982). In people with Borrelia infection, CSF IgM is persistently produced and the IgM index is a better indicator of this disease than is the IgG index (Fishman, 1992). Anti-West Nile Virus (WNV) IgM antibody production appears to occur intrathecally in horses and detection of CSF anti-WNV IgM may be used to differentiate previously vaccinated horses versus infected horses (Porter et al., 2004) Further studies need to be done in animals to determine the sensitivity and specificity of the various immunoglobulin alterations occurring in disease.

7. Electrophoretic patterns of csf protein in disease

Abnormalities in the CSF electrophoretic pattern can suggest categories of diseases (Sorjonen, 1987; Sorjonen et al., 1991). In one study, dogs with inflammatory diseases had one of three patterns: (1) little or no blood-brain barrier disturbance (as determined by CSF albumin concentration and the albumin quota) with decreased gamma globulin, (2) mild blood-brain barrier disturbance with markedly increased gamma globulin, and (3) moderate or marked blood-brain barrier disturbance with increased gamma globulin. Dogs with intervertebral disk protrusion or cervical spondylomyelopathy had a pattern of normal barrier function or severe barrier disturbance with decreased alpha globulin. Dogs with brain neoplasia had marked barrier disturbance and normal or mildly increased alpha and beta globulins (Sorjonen et al., 1991). However, a more recent study using high-resolution agarose electrophoresis was unable to differentiate various categories of neurological disease in dogs using this technique (Behr et al., 2006).

In the gamma globulin region, three patterns of protein bands can occur: monoclonal, oligoclonal, and polyclonal. Oligoclonal bands are associated with disease and are seen in a high percentage of people with multiple sclerosis or encephalitis. These bands, readily identifiable against the low background of normal polyclonal IgG in the CSF, are thought to represent the products of a limited number of plasma cell clones. Oligoclonal bands unique to CSF (i.e., not present in serum) indicate intrathecal synthesis of immunoglobulin and may be more sensitive than the IgG index in detecting this synthesis. People with multiple sclerosis may have a normal IgG index yet have CSF oligoclonal banding; thus, the demonstration of these bands is considered by some to be the single most useful test in the diagnosis of multiple sclerosis (Kjeldsberg and Knight, 1993). Oligoclonal bands are also seen in patients with inflammatory diseases and in some patients with neoplasia (Fishman, 1992). Occasionally, a single (monoclonal) band is identified in the CSF electrophoretic pattern of people. Monoclonal bands have been seen in neurologically normal people as well as in patients with neurological disease (Kjeldsberg and Knight, 1993).

8. Other csf proteins

Numerous attempts have been made to correlate specific CSF proteins, particularly “brain-specific” proteins, with specific diseases. Proteins such as C-reactive protein, interferon, myelin basic protein, and S-100 are increased in the CSF associated with neurological disease, but these increases are found in many heterogeneous conditions. This nonspecificity limits the clinical utility of many of these specific protein assays. However, the measurement of some of these proteins is thought to be useful as a screening procedure for neurological disease or as an indication of prognosis (Fishman, 1992; Kjeldsberg and Knight, 1993; Lowenthal et al., 1984). Immunoassay detection in the CSF of the brain-derived protein 14-3-3 appears to be helpful for the diagnosis of transmissible spongiform encephalopathies in both animals and people (Hsich et al., 1996; Sanchez-Juan et al., 2006). An autoantibody against canine glial fibrillary acidic protein present in astrocytes has been detected in the CSF of two pug dogs affected with necrotizing encephalitis (Uchida et al., 1999). However, it is unknown if the presence of this antibody is a primary or secondary phenomenon.

1. Bacterial Diseases

In central nervous system aerobic or anaerobic bacterial infections, the CSF may be clear, hazy, or turbid (depending on the cell count), and colorless or amber with moderate to marked elevations of total white blood cell count and total protein concentration. Because of the elevated protein concentration, the CSF may clot or foam when shaken. The white blood cell differential count characteristically has a high percentage of neutrophils (>75%), which may be degenerate (Baum, 1994; Dow et al., 1988; Foreman and Santschi, 1989; Green and Smith, 1992; Kornegay, 1981; Meric, 1988; Rand et al., 1994b; Santschi and Foreman, 1989; Scott, 1995; Sturges et al., 2006; Tipold, 1995). The protein is composed of albumin that has crossed the diseased blood-brain/CSF barrier and immunoglobulin produced intrathecally; therefore, the IgG index is usually elevated (Tipold, Pfister, & Vandevelde (1993b), Tipold, Pfister, Zurbriggen, & Vandevelde (1994)). The IgM and IgA levels may be normal or increased (Tipold et al., 1994). The CSF of animals with chronic or treated bacterial infections may be nonsuppurative with mild to moderate elevations of total white blood cell count and total protein concentration (Green and Smith, 1992; Sturges et al., 2006). Occasionally extracellular or intracellular bacteria may be seen, either on a routine Wright's stain or a Gram stain (Foreman and Santschi, 1989; Green and Smith, 1992; Kornegay, 1981). Because prior antibiotic therapy is common, and some bacteria undergo rapid autolysis in the test tube, bacterial culture of these infections is often unrewarding. Nonetheless, culture should be attempted. Polymerase chain reaction techniques may be used to detect the presence of bacterial DNA (Finno et al., 2006; Peters et al., 1995).

2. Viral Diseases

The CSF associated with viral diseases is characterized by nonsuppurative inflammatory changes. The total white blood cell count and total protein are generally mildly to moderately elevated. The white cell population may be mixed with a majority of mononuclear cells or may be entirely mononuclear cells. Occasionally, neutrophils predominate, particularly in the early stages of disease or in certain diseases (discussed later). The IgG index is commonly elevated (Bichsel et al., 1984b; Tipold et al., 1994). The IgA and IgM levels may also be elevated. The CSF of viral infections may also be normal, particularly if the meninges or ependyma is not involved (Fankhauser, 1962; Fishman, 1992; Rand et al., 1994b; Tipold, 1995; Tipold et al., 1994).

1. Fibrocartilaginous embolism

The CSF characteristics associated with fibrocartilaginous embolism (FCE) are reported for dogs and horses. About one-third of the reported canine cases have normal CSF, about one-third have a mononuclear pleocytosis and increased protein, and about one-third have albuminocytological dissociation (Bichsel, Vandevelde, & Lang (1984a), Bichsel, Vandevelde, Vandevelde, Affolter, & Pfister (1984b); Cauzinille and Kornegay, 1996; Gandini et al., 2003; Grunenfelder et al., 2005). Similar findings are reported for two horses (Jackson et al., 1995; Taylor et al., 1977). However, we have seen marked neutrophilic pleocytoses associated with (histopathologically confirmed) severe, acute FCE in dogs (unpublished observation). The type of pleocytosis, neutrophilic or mononuclear, probably depends on the size, location, and age of the infarct. The CSF albumin is reported to be normal, and the IgG index either normal or slightly elevated, the latter possibly reflecting the subsequent inflammation around the lesion (Bichsel et al., 1984b; Tipold et al., 1993b). Dogs with normal CSF may have a better prognosis for recovery (Cauzinille and Kornegay, 1996; Gandini et al., 2003).

1. intracranial Intra-arachnoid cysts

Although several reports describe intracranial intra-arachnoid cysts (ICIACs) in dogs (Duque et al., 2005; Kitagawa et al., 2003; Saito et al., 2001; Vernau et al. (1997), Vernau et al. (2002); Von Kurnatowski et al., 2006), most do not report the CSF findings present with this condition. Several dogs with ICIACs are diagnosed with concurrent inflammatory brain disease (Duque et al., 2005; Kitagawa et al., 2003; Vernau et al., 1997; Von Kurnatowski et al., 2006), and some authors believe that ICIACs may therefore be incidental in some animals. One report describes two dogs with ICIACs with intracystic hemorrhage. One of these dogs had a normal CSF nucleated cell count but had some degenerate red blood cells and a moderate elevation of protein. The other dog had a mild mononuclear pleocytosis, a mild elevation of protein, and there was evidence of erythrocytophagia (Vernau et al., 2002).

2. Neosporosis, toxoplasmosis

Both Neospora and Toxoplasma can invade the central nervous system causing necrosis, vasculitis and a multifocal, granulomatous meningoencephalomyelitis. Neospora seems to have more of a predilection for the central nervous system than Toxoplasma, particularly in young dogs (Dubey, Carpenter, Speer, Topper, & Uggla (1988), Dubey, Carpenter, Topper, & Uggla (1989)). The CSF associated with neural protozoal infections generally has a mild to moderate increase in white blood cell count and total protein. Typically, the white blood cell differential count shows a mixed pleocytosis with monocytes, lymphocytes, neutrophils, and eosinophils in order of decreasing percentage (Averill and DeLahunta, 1971; Cuddon et al., 1992; Dubey, 1990a; Hass et al., 1989; Kornegay, 1981; Rand et al., 1994b; Tipold, 1995; Vandevelde and Spano, 1977). However, we have also seen marked pleocytosis with marked eosinophil predominance in histopathologically confirmed canine CNS neosporosis (W. Vernau, unpublished observation). Occasionally the white blood cell count and protein are normal (Parish et al., 1987; Tipold, 1995). The CSF IgG index was elevated in three of three dogs studied; in two of two dogs, the IgM was normal and the IgA was elevated (Tipold, Pfister, & Vandevelde (1993b), Tipold, Pfister, Zurbriggen, & Vandevelde (1994)). In a study of experimentally infected cats, T. gondii-specific IgG was intrathecally produced, but T. gondii-specific IgM was not detected (Munana et al., 1995). Antiprotozoal antibodies in the CSF may be detected by a variety of methods (Cole et al., 1993; Patton et al., 1991; Ruehlmann et al., 1995). However, the presence of antibodies does not necessarily indicate clinical disease (Dubey and Lindsay, 1993; Munana et al., 1995). Polymerase chain reaction techniques have been developed to identify the protozoa in tissue and fluids, including CSF (Novati et al., 1994; Parmley et al., 1992; Schatzberg et al., 2003; Stiles et al., 1996). Occasionally the organisms themselves may be seen in CSF cells (Dubey, 1990a; Gaitero et al., 2006; McGlennon et al., 1990).

In considering the CSF abnormalities of toxoplasmosis and neosporosis, two issues must be kept in mind. First, reports of toxoplasmosis before 1988 (when Neospora was identified) must be carefully scrutinized because many of these cases were actually neosporosis. Second, because T. gondii is not a primary pathogen, clinical toxoplasmosis is relatively rare and is seen mostly in conjunction with a second disease, particularly canine distemper, which may itself alter the CSF (Dubey et al., 1989).