Introduction

Cerebrospinal fluid (CSF) analysis^{5, 7–10, 13–15, 17, 18, 20, 22, 31, 32, 36, 37, 51, 64, 84, 85, 93, 96, 97, 104, 110}

1. Indications for CSF collection (CSF “tap”)
   
   
   
   1. Encephalopathies often are an indication for CSF analysis. In particular, infectious and noninfectious inflammatory diseases can be best characterized by evaluating the cellularity and protein levels in the CSF. Different inflammatory diseases lead to an accumulation of different types of cells in the CSF, and variably affect the amount and type of protein that is present. Degenerative, metabolic, traumatic, and neoplastic brain lesions may also alter the normal CSF. Any disease affecting the brain, including seizure disorders, should lead the clinician to consider CSF analysis as part of the diagnostic plan.
      
   2. Any spinal cord lesion, or myelopathy, that is not readily diagnosed on spinal imaging, should be evaluated by CSF analysis. Focal, multifocal, and diffuse spinal cord lesions will lead to changes in the CSF. Cerebrospinal fluid collection should be done prior to myelography, since myelographic contrast agents will change the character of the CSF, and will frequently produce a mild inflammatory response. These changes are believed to influence CSF analysis for at least three to five days following the myelogram. In one study of normal dogs, the CSF white blood cell (WBC) count returned to normal within 72 hrs of myelography, and CSF protein elevation resolved by seven days post-myelography.
      
   3. Lesions that affect the spinal nerve roots (radiculopathies) may be evaluated with CSF analysis. The meninges enclose the nerve roots distally until they become the peripheral nerves. Thus, any disease, especially one that is inflammatory in nature, that affects the spinal nerve roots may alter the CSF.
   
   
2. Relative contraindications and risks of performing a CSF tap
   
   
   
   1. Elevated intracranial pressure (ICP) due to mass lesions or inflammatory disease increases the risk of brain herniation (and subsequent death) when a CSF tap is performed. Typically, this is herniation of the cerebellar vermis and brain stem caudally through the foramen magnum. Cerebral herniation past the osseous tentorium caudally, or past the falx cerebri laterally, may also occur. The incidence of brain herniation following a CSF tap has been found to be slightly higher in cats than in dogs. Mannitol, corticosteroids, and hyperventilation may be used to decrease the risk. Because of the increased risk of herniation, CT or MRI should be performed prior to a CSF tap when mass lesions are suspected. It is unknown whether there is any advantage of lumbar versus cerebellomedullary cisternal CSF collection in terms of brain herniation risk.
      
   2. Inadvertent penetration of the parenchyma at the cerebellomedullary angle may lead to temporary signs of brain-stem disease, such as vestibular abnormalities, or cessation of voluntary respiration and death. The vestibular dysfunction may take a long time to subside or be permanent.
      
   3. Performing CSF collection in small animals requires general anesthesia, as a rule. If the patient is an unacceptable anesthetic risk, lumbar puncture may be attempted with sedation and local (epidural) anesthesia.
   
   
3. Areas of CSF procurement and collection technique
   
   
   
   1. 
      

      One milliliter per 5 kg body weight of CSF can be safely removed at one time for analysis. Usually, 1 to 1.5 ml are collected (about ten drops). The fluid should be collected in a sterile glass tube, preferably without EDTA (e.g. red-top tube). EDTA may cause falsely elevated protein concentrations, as well as falsely low cell concentrations in small samples. Since EDTA is bactericidal, it may interfere with CSF culture results in cases of CNS bacterial infections. However, EDTA may help preserve cellular morphology. The authors routinely collect both a red-top and purple-top (EDTA) sample for analysis.

      
      

      CSF is most commonly obtained from the cerebellomedullary cistern (cisternal tap; Fig. 5.1). CSF collected from this site may be more representative of lesions involving the brain than CSF collected from a lumbar puncture. Anatomic landmarks useful in performing cisternal CSF taps include the external occipital protuberance, the cranial aspect of the dorsal spine of the axis (C2 cervical vertebra), and the transverse processes (“wings”) of the atlas (C1 cervical vertebra). The patient is placed in lateral recumbency and the neck is flexed by an assistant. The animal’s nose must be kept parallel with the table. A noncollapsing endotracheal tube should be used, to avoid occluding airflow during the procedure. Red rubber endotracheal tubes should be avoided, because it has been shown that these tubes are much more prone to complete kinking with neck flexion when compared with polyvinyl chloride (PVC) endotracheal tubes. The assistant should “tuck in” the animal’s chin and push the external occipital protuberance toward the individual performing the tap. Placing some form of support under the neck (e.g. rolled-up paper towel) will help keep the spine of the axis and the external occipital protuberance in line.

      

      
      

      The skin in the region of the tap is shaved and aseptically prepared, and a 22-gauge spinal needle with a stylet (20-gauge is acceptable in larger patients) is inserted on midline, directed toward the occipitoatlantal space. Sterile gloves are worn for the duration of the procedure. The proper location for needle insertion can be estimated in several ways. The authors prefer to locate the cranial aspect of the C2 spine with an index finger, then press firmly with the fingertip as the finger is simultaneously advanced cranially. In most patients, a ridge or “divot” can be palpated approximately one-third of the distance between the cranial aspect of the C2 spine and the external occipital protuberance. This ridge is the cranial aspect of the arch of C1. Inserting the needle just cranial to the ridge should allow entry into the occipitoatlantal space. An alternative method is to draw an imaginary line across the cranial limits of the wings of C1 and a perpendicular line from the external occipital protuberance caudally. The needle can be inserted at the intersection of these lines. The skin is punctured first, then the index finger and thumb of one hand (left hand for a right-handed person) is used to stabilize the needle against the skin surface, as the other hand is used to slowly advance the spinal needle. After every few millimeters of advancement, the stylet is removed to observe for CSF flow. Typically, the clinician will be able to feel the needle pass through fibrous tissue planes, producing a “popping” sensation. If the needle abuts bone, slight cranial or caudal redirection of the needle tip may allow entry into the dorsal subarachnoid space.

      
      
   2. 
      

      Lumbar puncture for CSF collection (lumbar tap) is usually performed at the L4/L5 space in large dogs or at the L5/L6 space in smaller dogs and cats (Fig. 5.2). Lumbar CSF may be more representative of lesions involving the thoracolumbar spinal cord than CSF from a cisternal puncture. The patient is placed in lateral recumbency and an area is shaved and aseptically prepared for CSF collection. The patient’s pelvic limbs are advanced cranially, in order to open up the interarcuate space. The authors prefer to face the ventral aspect of the patient, and bend over the patient to insert the spinal needle. The spinal needle is inserted just lateral to midline, adjacent to the caudodorsal limit of a spinous process (L6 for L5/L6 puncture; L5 for L4/L5 puncture). The needle is inserted at a 30–60° angle from an imaginary line drawn perpendicular to the long axis of the spine.

      

      
      

      After the interarcuate space is entered, the needle will pass through the dorsal dura mater. Often, at this point, a twitch of the pelvic limbs and/or tail will be noted. The needle is advanced to the floor of the vertebral canal, and the stylet is withdrawn. CSF is allowed to drip into a collection tube. Although the spinal needle penetrates the spinal cord during a lumbar CSF tap, this does not appear to cause any clinical problems.

      
      
   3. Collection site: CSF collection should be performed close to the location of the lesion or caudal to it. For brain and cervical diseases the cerebellomedullary region is appropriate. For thoracolumbar or lumbar lesions, CSF should preferably be collected in the lumbar region. In cases of multifocal disease, collection at both sites can be very valuable and is recommended.
   
   
4. 
   

   CSF evaluation

   
   

   There are a variety of tests that can be performed on CSF (Box 5.1). Typically, a total cell count, differential cell count (after cytocentrifugation), and a protein level are ascertained. A glucose level is occasionally obtained, and is normally 60–80% of the blood glucose level. If infectious disease is suspected, appropriate cultures or serology can be performed on the fluid. Electrophoresis of CSF may help to characterize the type(s) of protein present in the CSF. For some diseases (e.g. canine distemper virus, FIP [coronavirus] CNS infection in cats), amplification of genetic material via polymerase chain reaction (PCR) may be indicated. When lymphoma is suspected (in cases of lymphocytic pleocytosis or possible neoplastic lymphocytes in the CSF), flow cytometry, or PCR for lymphocyte antigen receptor rearrangement (PARR) can be used to confirm the diagnosis. Testing for PARR requires a larger volume and higher concentration of lymphocytes than what is required for flow cytometry. Ideally, cell counts should be performed within 30 min of CSF collection; however, there is evidence that reliable cell counts may be obtained up to 48 hrs later when the CSF is preserved through the addition of autologous serum. Prior treatment may alter the expected results of CSF analysis, especially in patients with inflammatory disease treated with corticosteroids.

   
   
   
   
   1. 
      

      Color and clarity

      
      

      Normal CSF is clear and colorless, with the consistency of water. Prior hemorrhage (occurring a minimum of 10 hrs prior to CSF collection) in the CSF may result in a yellow tinge, referred to as xanthochromia. This discoloration can persist for 2–4 wks following hemorrhage into the subarachnoid space, but is usually resolved by four to eight days. Other potential causes for xanthochromia are severe icterus, and markedly elevated CSF protein levels.

      
      

      Gross blood contamination may be iatrogenic, or due to ongoing hemorrhage in the subarachnoid space. Iatrogenic hemorrhage is more common with lumbar taps, compared to cisternal taps. Although iatrogenic hemorrhage interferes with interpretation of CSF results, the extent to which it does so is controversial. It has been suggested that each 500 red blood cells (RBCs)/ml in a hemorrhagic CSF tap may account for one white blood cell (WBC)/ml in dogs, each 100 RBCs/ml accounting for one WBC/ml in cats. However, it has also been demonstrated that RBC counts in CSF as high as 15,000/ml can occur with minimal elevation of the WBC count. The effect of hemorrhage on CSF protein levels is typically low, with approximately 1200 RBCs/ml needed to increase the protein concentration by 1 mg/dl.

      
      

      Increased turbidity of CSF is usually due to an elevated number of cells (over 200 WBCs/ml, over 400 RBCs/ml), and occasionally due to increased protein levels. Elevated protein levels in CSF will also cause the fluid to be more viscous. CSF that tends to clot is rare, and is caused by markedly increased amounts of protein.

      
      
   2. 
      

      Total and differential WBC count

      
      

      Though the actual number may vary with the laboratory used, there are typically fewer than five nucleated cells/ml of CSF. In normal dogs and cats, lumbar CSF typically has fewer WBCs/ml than cisternal CSF. The distribution should be predominantly mononuclear cells with only occasional neutrophils.

      
      
   3. 
      

      Protein level

      
      

      Quantitative determinations are the most accurate. Although each laboratory will establish normal ranges, normal protein concentration for cisternal CSF is less than 27 mg/dl in dogs and cats. Normal protein levels will be higher when the CSF is collected from a lumbar puncture (approximately twice that of cisternal CSF, or less than 45 mg/dl).

   
   
5. CSF in disease
   
   
   
   1. The greater the meningeal or ependymal involvement, in general, the greater the number of WBCs expected in the CSF. Deep-seated parenchymal lesions may be associated with mildly increased or normal cell counts, often with elevated protein levels. Increased nucleated cell counts in the CSF are referred to as pleocytosis. A normal cell count with an elevated protein level is often referred to as albuminocytologic dissociation.
      
   2. Suppurative means a predominance of neutrophils. A neutrophilic pleocytosis is often associated with bacterial infections and corticosteroid-responsive (aseptic) meningitis. Other diseases in which neutrophils may predominate in CSF include some viral encephalitides (e.g. acute canine distemper infection, FIP meningoencephalitis in cats), fungal infections, meningiomas, and fibrocartilaginous embolic myelopathy (FCEM). Neutrophils associated with infectious diseases (e.g. bacterial meningoencephalitis) are more likely to be degenerate than those that occur in noninfectious (e.g. corticosteroid-responsive meningitis) disorders.
      
   3. Mononuclear cell pleocytosis refers to a predominance of either lymphocytes or macrophages in the CSF. This is the most common pleocytosis encountered, and is usually associated with granulomatous meningoencephalomyelitis (GME) in dogs. The necrotizing encephalitides (Pug/Maltese encephalitis, Yorkshire Terrier encephalitis) are usually characterized by primarily lymphocytic pleocytosis. Lymphosarcoma involving the CNS may also be associated with a lymphocytic pleocytosis. A predominantly mononuclear pleocytosis can be caused by fungal, viral (e.g. canine distemper virus), protozoal, rickettsial, and chronic bacterial infections.
      
   4. Eosinophilic pleocytosis is rare. It has been associated with aberrant parasite migration in the CNS, rabies virus, and cryptococcal, protozoal, and protothecal infections. There is also a rare idiopathic condition called eosinophilic meningoencephalitis that is characterized by a substantial proportion of eosinophils in the CSF.

Neuroimaging^{25–27, 34, 44, 49, 50, 52, 58, 75, 86, 88, 102, 103, 107}

1. Survey (“plain”) radiographs^{6, 57, 61, 76, 79, 91}
   
   
   
   1. The main use of survey radiography is as a rapid screening tool for obvious bony abnormalities. Soft tissue structures of the CNS are poorly visualized, if at all, with survey radiographs. A minimum of two views (e.g. lateral and ventrodorsal) is required.
      
   2. Survey radiographs of the skull may reveal fractures and osseous neoplasia, or may suggest soft tissue or fluid densities in the nasal passages, sinuses, or middle ear canals. Appropriate positioning for skull radiographs requires general anesthesia. In general, CT is preferable to radiographs for most of these purposes.
      
   3. 
      

      Survey spinal radiographs in the unanesthetized patient are often of questionable diagnostic quality due to poor patient positioning and patient movement. Obvious osseous tumors (Fig. 5.3), advanced discospondylitis (Fig. 5.4), displaced fractures or luxations (Fig. 5.5), and congenital vertebral anomalies (e.g. hemivertebrae), may be appreciated on survey spinal films in the unanesthetized patient. If the patient is likely to undergo anesthesia for myelography and/or surgery (e.g. acutely paralyzed Dachshund), performing survey radiographs on the unanesthetized patient is usually not justifiable.

      

      

      

      
      
   4. High-quality spinal radiographs under anesthesia may reveal a number of abnormalities. In patients with intervertebral disc disease, collapsed disc spaces, decreased size of the intervertebral foraminae, or mineralized disc material within the vertebral canal or intervertebral foraminae may be appreciated (Fig. 5.6). Subtle vertebral fractures or subluxations with minimal displacement may be more readily apparent on radiographs performed with the patient anesthetized versus radiographs procured when the patient is awake. Other radiographic abnormalities that may be more evident in the anesthetized patient include subtle bony lesions associated with neoplasia (e.g. mild bone lysis), abnormalities associated with articular facets (including small fractures), and subtle bony changes suggestive of early discospondylitis (e.g. small regions of vertebral endplate lysis). In general, survey radiography is less sensitive than CT for identifying subtle bone lysis and small fractures.

      

   
   
2. Myelography^{11, 24, 40, 60, 62, 68, 70, 89, 108, 109}
   
   
   
   1. Myelography is a procedure in which spinal radiographs are obtained following the injection of a radiopaque contrast agent into the subarachnoid space. Nonionic, iodinated, water-soluble contrast agents are used, iohexol and iopamidol being the most common.
      
   2. The myelogram is performed via a cisternal or lumbar tap. CSF should be collected prior to contrast injection, as the contrast will change the composition of the CSF and may prevent accurate analysis for a minimum of three to five days. The authors prefer lumbar versus cisternal injection of contrast, regardless of the area of interest. Lumbar myelography often results in better image quality and is safer than cisternal myelography. The dosage of contrast used for a regional study (e.g. cervical myelogram with cisternal contrast injection) is 0.3 ml/kg body weight. For full studies (e.g. cervical myelogram with lumbar injection), the dose is 0.45 ml/kg body weight. The contrast is injected slowly, approximately 2–3 ml per minute. The authors prefer to administer a test injection (e.g. 0.5–1.0 ml, depending on patient size) to ensure the contrast is in the subarachnoid space, before administering the remainder of the calculated contrast dose.
      
   3. Myelography is used to assist in the diagnosis of myelopathies. It is indicated for cases in which survey radiographs are either normal or inconclusive, despite neurologic evidence of myelopathy. Myelography is also often helpful in estimating the location, extent, and severity of spinal lesions. The ability to easily and rapidly visualize the entire spinal cord is an advantage of myelography over other imaging procedures (e.g. CT, MRI). Myelography is also generally less costly and may be more readily available than CT or MRI in certain situations (e.g. emergencies). As more veterinary referral centers acquire high-field MRI systems, myelography and other contrast radiographic procedures (e.g. epidurography, discography) are assuming a less important role in the neuroimaging of dogs and cats.
      
   4. 
      

      Despite the advantages of myelography, it is a somewhat invasive procedure and is associated with a low level of inherent risks. Overall, postmyelographic seizures occur in approximately 3–20% of dogs undergoing the procedure; this adverse event is more likely to occur in larger dogs (more than 20 kg), and is also more likely to occur with cisternal vs. lumbar contrast injections. In addition, the likelihood of postmyelographic seizure activity increases with increasing total volume (not dose on a ml/kg basis) of contrast injected. This is the main factor explaining a significant difference in the prevalence of seizures in two main studies. In a study with 503 dogs, the authors found a 3% prevalence of seizures. The mean volume of iohexol was 11.7 ml and 4.5 ml in dogs that did have and did not have seizures, respectively. In another study where the authors found a prevalence of 21.4% of seizures in 182 dogs, the mean volume was 16.8 ml and 9.1 ml for dogs that did and did not have seizures. Since the total volume of contrast medium is the main factor causing postmyelographic seizures, it is recommended to limit the initial volume of contrast to 8 ml and only use larger volumes if necessary. Larger dogs, primarily those with cervical lesions, also have a higher risk of seizures. Most patients that seizure will do so only once or twice in the 24 hrs following the procedure, and the seizures usually cease with intravenous diazepam injection (0.2–0.4 mg/kg). Maintaining slight elevation of the patient’s head during and after the procedure (until the patient is awake) and assuring hydration with intravenous fluids during and 24 hrs after myelography are recommendations to limit the occurrence and severity of postmyelographic seizure activity.

      
      

      All dogs receiving a myelogram should be under close hospital observation (e.g. in an ICU) for the first 24 hrs following the procedure. Parenchymal damage from insertion of the needle is rare in myelography, but may occur, especially in the cervical region. Worsened neurologic status post myelogram is usually caused by transient chemical myelitis secondary to contrast injection. The risk for this may be higher in patients with existing inflammatory disease or chronic spinal cord compression (e.g. chronic type II disc disease). In the authors’ experience, the risk of transient neurologic worsening appears to be highest in dogs with cervical spondylomyelopathy (CSM). These dogs may be markedly worse the day after the myelogram, but typically regain premyelogram neurologic status within 72 hrs. Inadvertent contrast injection into the parenchyma or the central canal of the spinal cord may cause worsened neurologic status. In most cases, patients recover from this iatrogenic trauma, but permanent deficits may occur in a small proportion of animals. Myelography is contraindicated for patients with known or highly suspected inflammatory disease of the CNS, as it may cause worsening of neurologic status.

      
      

      Myelography is also contraindicated in patients that may have elevated ICP. Since cervical hyperesthesia occasionally is associated with forebrain lesions, any historical or clinical indication of an underlying encephalopathy should prompt consideration of an alternative imaging modality (e.g. CT or MRI).

      
      
   5. 
      

      There are four basic myelographic patterns: normal, extradural, intradural/extramedullary, and intra-medullary (Fig. 5.7). Normally, the contrast columns parallel each other and conform to the vertebral canal, except at the cauda equina region, where the subarachnoid space tapers. The spinal cord ends at about the L6 vertebral region in most dogs and at the first sacral vertebral region in most cats, although there may be quite a bit of variation between breeds.

      

      
      

      The spinal cord is normally larger at the cervical and lumbosacral intumescences. The ventral subarachnoid space is often less prominent than the dorsal subarachnoid space in the thoracolumbar region in dogs. The dorsal subarachnoid space in the atlantoaxial region is often wider than the remainder of the spinal cord. The cervical spinal cord region in cats often appears wider on myelography, in comparison to dogs. A normal myelographic pattern is often associated with degenerative myelopathy and FCEM. A normal myelogram may also occur with inflammatory myelopathies.

      
      

      Intervertebral disc extrusion/protrusion is the most common cause of an extradural myelographic pattern. Other causes of extradural patterns include vertebral fracture/luxation, congenital vertebral anomalies, hypertrophied soft tissue structures (e.g. interarcuate ligament, synovial membranes), extradural hemorrhage, vertebral neoplasia, and soft tissue neoplasia (e.g. feline lymphosarcoma). It is important to realize that the nature of an extradural compression is best appreciated when viewed tangential to the direction of the cord deviation. For example, if a disc extrusion is compressing the cord from ventral to dorsal, with no lateralizing component, the myelographic pattern as viewed from a ventrodorsal view (parallel with direction of compression) could be misinterpreted as intramedullary. With intervertebral disc extrusions, which are often ventrolateral (i.e. ventral but somewhat lateralized), it is usually helpful to obtain oblique views in addition to standard dorsal and ventral views, in order to ascertain the correct side of disc extrusion for purposes of surgical planning. In one study, the accuracy of correctly identifying the side of disc extrusion was significantly higher for oblique vs. ventral views, but the accuracy was higher still when information from these views was combined.

      
      

      An intradural/extramedullary pattern is produced when there is a lesion within the subarachnoid space (intradural), but not invading the parenchyma of the cord (extramedullary). As the contrast flows around the obstructive lesion, it may be outlined, appearing as a “filling defect.” Sometimes, the filling defect is incompletely outlined, and resembles a golf tee, hence the term “golf tee sign” (Fig. 5.8). Intradural/extramedullary patterns are most often associated with neoplasia, primarily meningiomas and nerve sheath tumors. Much less commonly, intradural hemorrhage may lead to this myelographic pattern. Intradural/extramedullary lesions may produce enough spinal cord swelling that contrast is excluded from the region of the mass. In such cases, the myelographic pattern may appear to be intramedullary. In such cases, a CT is often performed through the abnormal region, as contrast is better visualized on CT images.

      

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