Cerebrospinal fluid and blood lactate concentrations as prognostic biomarkers in dogs with meningoencephalitis of unknown origin

Journal Pre-proof Cerebrospinal fluid and blood lactate concentrations as prognostic biomarkers in dogs with meningoencephalitis of unknown origin ´ M. Portero, E. Mart´ınez de Merlo, C. Perez, M. Benito, M.A. Daza, C. Fragio

PII:

S1090-0233(19)30129-7

DOI:

https://doi.org/10.1016/j.tvjl.2019.105395

Reference:

YTVJL 105395

To appear in:

The Veterinary Journal

Accepted Date:

10 October 2019

´ Please cite this article as: Portero M, de Merlo EM, Perez C, Benito M, Daza MA, Fragio C, Cerebrospinal fluid and blood lactate concentrations as prognostic biomarkers in dogs with meningoencephalitis of unknown origin, The Veterinary Journal (2019), doi: https://doi.org/10.1016/j.tvjl.2019.105395

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Original Article Cerebrospinal fluid and blood lactate concentrations as prognostic biomarkers in dogs with meningoencephalitis of unknown origin M. Portero a, *, E. Martínez de Merlo a, C. Pérez a; M. Benito a, M.A. Daza a, C. Fragio a a

Department of Small Animal Medicine and Surgery, Hospital Clinico Veterinario Complutense, Universidad Complutense, Madrid, Avenida Puerta de Hierro s/n. 28040 Spain *Corresponding author. Tel.: +0034 913943812. E-mail address: [email protected] (M. Portero).

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Highlights Prognostic factors in meningoencephalitis of unknown origin (MUO) in dogs are controversial.

Lactate is a prognostic biomarker in many diseases and its concentration can be determined in cerebrospinal fluid (CSF).

High brain metabolic activity (inflammation) or raised intracranial pressure

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increased CSF lactate levels.

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CSF lactate >4 mmol/L in MUO in dogs could be associated with lower

Abstract

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survival.

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Meningoencephalitis of unknown origin (MUO) is a common inflammatory disease

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of the central nervous system. Several studies investigated finding prognostic factors, but results are contradictory. The aim of this study was to determine the concentrations of blood lactate (Blood-L) and cerebrospinal fluid lactate (CSF-L) in dogs with MUO for prognostic purposes. A total of 45 dogs with MUO (MUO group) and 11 with idiopathic epilepsy (IE group) were included. In the MUO group, 22 dogs were treated with

prednisolone + cytosine arabinoside, 17 with prednisolone ± cyclosporine, and six received no treatment.

In the MUO group, there was a strong-moderate positive correlation between BloodL and CSF-L (= 0.63557; P< 0.0001), a strong-moderate negative correlation between survival and CSF-L (= -0.50210; P< 0.0004), and a weak negative correlation between survival and Blood-L (= -0.35685; P< 0.0220). Dogs with a favourable response to

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treatment at 1 month had lower initial concentrations of Blood-L and CSF-L (P< 0.0010; P< 0.0037), and those with a worse response had higher values (P< 0.0497; P< 0.0004).

Dogs that remained stable with treatment showed lower CSF-L concentrations (P<

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0.0013). Dogs with Blood-L>4 mmol/L (P< 0.03) and/or CSF-L> 4 mmol/L (P< 0.009)

had lower survival rates with the latter also showing more severe signs, probably

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indicating severe neuronal damage. These findings suggest that concentrations of CSF-L

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and Blood-L in dogs with MUO could be used as prognostic indicators.

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Keywords: Canine; Cerebrospinal fluid; Lactate; Meningoencephalitis of unknown origin; Prognosis

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Introduction

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The incidence of non-infectious inflammatory diseases of the central nervous system (CNS) in dogs is increasing, especially granulomatous meningoencephalitis, necrotizing meningoencephalitis and necrotizing leukoencephalitis, where a definitive diagnosis is histopathological (Coates and Jeffery, 2014). It is very rare to obtain histopathological confirmation ante-mortem, so the term meningoencephalitis of unknown origin (MUO) has been created to encompass cases with clinical signs and

magnetic resonance and/or cerebrospinal fluid (CSF) findings compatible with noninfectious inflammatory disease of the CNS but with no histopathological confirmation (Talarico and Schatzberg, 2010). Increasing numbers of studies focus on finding prognostic factors in MUO but results are contradictory. However, there seems to be a consensus that decreased mentation is a negative prognostic factor (Coates et al., 2007; Cornelis et al., 2016[a]; Cornelis et al., 2016[b]).

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Lactate is one of the major prognostic biomarkers in several disease states, and its concentration can be determined in biological tissues and fluids. The brain has robust

metabolic activity and depends on the constant supply of glucose and oxygen. As activity

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increases, increased metabolic energy is required in neurons which is achieved by glucose

oxidation with the consequent formation of pyruvate and lactate (Smith et al., 2003). Any

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changes associated with poor brain oxygenation, high metabolic activity or raised

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intracranial pressure may increase lactate concentration in the CSF (CSF-L; Ronquist et al., 1985).

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The aim of this study was to establish whether lactate concentrations, in both

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blood and CSF, can be useful in determining prognosis of dogs with MUO.

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Materials and methods MUO group

A prospective clinical study between March 2010 and April 2017 was conducted

with dogs diagnosed with MUO based on criteria established in the meta-analysis of Gragner et al. (2010) as part of the normal clinical diagnostic protocol. Informed consent was obtained from all dog owners. The inclusion criteria for the MUO group were (1)

animals older than 6 months; (2) no alterations in physical examination and no concomitant diseases at the time of the diagnosis; (3) blood analytical parameters within the reference ranges (complete blood count, total solids, urea, creatinine, alanine aminotransferase, and glucose); (4) negative serum antibody titres to Leishmania infantum and Ehrlichia canis by an indirect fluorescence antibody test (5) neurological examination compatible with focal or multifocal signs of CNS impairment; (6) changes in the CSF analysis (increase in proteins and/or pleocytosis) and/or

presence of

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hyperintense lesions in T2-Weighted magnetic resonance imaging compatible with MUO; (7) an absence of canine distemper virus, Leishmania infantum, Toxoplasma gondii, and Neospora caninum in CSF determined by PCR; (8) CSF- L measurement;

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(9) information on the dog’s progress at the end of the study; and (10) dogs with histopathological confirmation of MUO only need CSF-L measurement to be included in

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the study.

Control group

The control group was composed of dogs diagnosed with idiopathic epilepsy (IE

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group) according to the consensus of the International Veterinary Epilepsy Task Force (De Risio, 2015; Hülsmeyer et al., 2015; Potschka et al., 2015; Rusbridge et al., 2015)

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with no concomitant diseases at the time of the diagnosis and absence of epileptic seizures

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15 days prior to diagnosis and CSF-L measurement. 15 days prior to diagnosis and CSF-L measurement.

Blood and CSF lactate analysis Magnetic resonance imaging (Panorama 0.23T (Philips Medical Systems) and a CSF tap collection were performed under general anaesthesia. Pulse sequences included

sagittal and transverse T2-weighted images (T2-WI); transverse T2-fluid-attenuated inversion recovery images (FLAIR); transverse Fast Field Echo tridimensional T2-weight images (FFE3D-T2*) and transverse T1-weighted images (T1-WI) before and after paramagnetic contrast injection (gadolinium 0.1 mmol/kg (Prohance Sol. 279.3 mg/mL, Laboratorios Rovi).

The CSF collection was obtained by cerebellomedullar cistern puncture with a 21-

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gauge half-inch spinal needle and 1.5-2 mL CSF was collected using dripping in EDTA tubes. For CSF analysis, total nucleated cell count (TNCC) total protein (TP) concentration and cytological differentiation were recorded as previously described (Di

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Terlizzi and Platt, 2009). CSF samples with blood contamination (red blood cell count

>4000 cells/μl) were excluded from the study owing to interference in determining CSF-

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L concentrations. Immediately following the CSF collection, CSF-L and blood lactate

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(Blood- L) concentrations were measured using the Accutrend Analyzer Roche portable lactate analyzer (Roche Diagnostic GmbH). Blood-L samples were drawn from the jugular vein into an EDTA tube. A fixed volume of 25 μl of CSF and blood in EDTA was

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used for lactate measurements in all dogs (Stevenson et al., 2007; Almeida et al., 2014).

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Treatment of dogs with MUO

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Dogs with MUO were treated with prednisolone (1 mg/kg q12h, gradually reduced every 4-6 weeks) alone or in combination with cytosine arabinoside (Citarabina 500 mg, Pfizer; 50 mg/m2 SC q12h for 48h, cycles every 3 weeks with the subsequent weekly increase every four cycles) or cyclosporine (Atopica 25 mg, Novartis; 7.5 mg/kg, once daily, gradually reduced every 4-6 weeks; Adamo and O’Brian., 2004; Lowrie et al., 2013).

Statistical methods All analyses were performed using Statistical Package for Social Science (SPSS) version 22 (SPSS) and SAS version 9.4 (SAS Institute). A descriptive analysis was performed including mean, standard deviation, maximum, minimum, and median of the quantitative variables. Comparisons between variables were determined using the Wilcoxon rank-sum test and the Spearman correlation coefficient (). Spearman

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correlations were classified as: strong-perfect (0.76-1.0), moderate-strong (0.51- 0.75), weak (0.26-0.5), and low (0-0.25). The overall survival time (calculated from the time of

diagnosis until the dog’s death or euthanasia, or the end of the study period) and the 7-

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day survival rate were evaluated using the Kaplan-Meier curves for all variables. For CSF-L and Blood- L, a cut-off point was set at 4 mmol/L (according to the maximum

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CSF-L concentration obtained in the IE group); for CSF TP the cut-off point was 0.3 g/L

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and for TNCC it was 100 cells/ μL. Statistical significance was set at P< 0.05.

Results

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Dogs in the MUO group and IE group

A total of 56 dogs were included, with 45 in the MUO group: there were 20

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females and 25 males with age at presentation of 6.6 ± 3.9 years and an average

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bodyweight of 7.3 ± 5.5 kg; breeds included Yorkshire terrier (n=12), crossbreeds (n=11), Bichon Frise (n=6), Pug (n=3), Maltese (n=3), Miniature Poodle (n=2), Pomeranian (n=2) and one each of Miniature Pinscher, Shih-Tzu, Brittany spaniel and Miniature Schnauzer. The IE group included 11 dogs, 10 males and one female with age at presentation of 4.7 ± 2.6 years and an average bodyweight of 23 ± 9.48 kg; breeds included crossbreeds (n=5), French bulldog (n=2), Labrador retriever (n=2), Alaska malamute (n=1) and

German shepherd dog (n=1). Although all animals had blood tests within the reference range, only blood values obtained less than 1 month before diagnosis were analysed. There were no statistically significant differences between breeds, sex or weight and the concentrations of CSF-L or blood-L before the study when the control group the MUO group were compared. Results of blood and CSF analyses are summarized in Table 1.

Treatment, outcome, and lactate concentrations

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Eighteen dogs in the MUO group had received previous treatment with glucocorticoids. However, at the time of the diagnosis, they had not received treatment

for at least 15 days. A total of 49% (n=22/45) of the dogs were treated after diagnosis

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with prednisolone + cytosine arabinoside (group A) and 38% (n=17/45) were treated with

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prednisolone (n=13/45) ± cyclosporine (n=4/45; group B). Thirteen percent (n=6/45; group C) did not receive specific MUO treatment. These dogs showed severe alteration

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in mental state (coma) were euthanased at the time of the diagnosis or in the following 24 h. The concentrations of Blood-L and CSF-L in the different treatment groups are shown

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in Table 2. There were no significant differences in the overall survival rate between the

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two treatment groups (A vs. B).

Regardless of the treatment administered, 67% of the treated dogs had favourable

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responses in the first 7 days. Results of blood and CSF parameters in dogs with survival <7 days or >7 days are summarized in Table 3.

All dogs in the IE group remained alive at the end of the study. The median survival time in the MUO group was 387 ± 70 days, regardless of the treatment given (in MUO treatment group A: 955 ± 569 days and in MUO treatment group B: 415 ± 234

days). In MUO group, 14/45 dogs (31.11%) were alive at the end of the study. Of the 31 dogs that died, six (19.35%) died from causes unattributable to MUO. Twenty percent of the dogs under treatment were considered cured (dogs with neurological examination findings within normal limits and which had been treatment-free for at least 1 year), 44% remained stable under treatment (dogs with improved clinical signs but that continued to show neurological abnormalities) and 36% showed an unfavourable response to treatment

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in the first month.

Of all the parameters analysed in blood and CSF common to the IE and MUO

groups, statistically significant differences were only found between these groups in TP

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concentrations in CSF (P< 0.0007), TNCC (P< 0.0001) and CSF-L (P< 0.01). In the MUO group, the following Spearman correlations () were found: a strong-moderate

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positive correlation between Blood-L and CSF-L (= 0.63557; P< 0.0001), a strong-

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moderate negative correlation between survival and CSF-L (= -0.50210; P< 0.0004),

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and a weak negative correlation between survival and Blood-L (= -0.35685; P< 0.0220).

In dogs that received treatment (groups A and B), statistically significant

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differences were demonstrated (Wilcoxon rank-sum test) in Blood-L and CSF-L concentrations in those showing a favourable response to treatment at 1 month (lower

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concentrations; P< 0.0037 and P< 0.001, respectively) and in those showing an unfavourable response (higher concentrations; P <0.0004 and P< 0.0497, respectively). The dogs in these two groups that remained stable under the treatment showed lower CSF-L concentrations (P <0.0013).

On analysing survival at 7 days of the diagnosis (Wilcoxon rank-sum test) in the three groups (A, B and C-untreated), statistically significant differences were found in Blood-L concentrations (P< 0.0001) and CSF-L (P< 0.0001). Dogs with elevated BloodL and CSF-L had lower survival rates. No differences were found in the remaining blood parameters evaluated or in CSF. On analysing the overall survival of all the dogs using the Kaplan-Meier survival curves, dogs with Blood-L and CSF-L >4 mmol/L showed significantly lower survival than those with lower values (P< 0.03 and P< 0.009,

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respectively; Figs. 1 and 2).

Discussion

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Variability in MUO clinical signs, diagnostic imaging findings, different choices

of treatment and response to treatment make it difficult to unify the prognostic factors in

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dogs with MUO.

This study is the first to evaluate Blood-L and CSF-L concentrations as prognostic biomarkers in this disease group. No abnormalities were observed in blood

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parameters evaluated in dogs in the MUO group, except for elevated Blood-L concentrations in some cases, although the differences found in Blood-L between the

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MUO and IE group were not statistically significant. When Blood-L values were analysed

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independently in dogs that were euthanased at the time of diagnosis (group C; usually because these dogs presented with a severe degree of the disease with alteration in mental state (coma)), hyperlactatemia was severe and reached mean values of 7±5.37 mmol/L (Table 2). A previous study by Cornelis et al., (2016[b]), included Blood-L concentrations with mean values of 2.1 mmol/L in the group that survived < 7 days and 1.4 mmol/L in the group that survived > 7 days; these differences were statistically significant

(P<0.026). In our study, we also found statistically significant differences in Blood-L concentrations for survival less than or greater than 7 days (P<0.0001); however, our mean values were 6.27 mmol/L in the group that survived < 7 days and 2.05 mmol/L in the group that survived > 7 days (Table 3). Hyperlactatemia in dogs with lower survival rates may be due to a hypermetabolic state and/or tissue hypoxia due to decreased ventilation which may be a consequence of MUO or any other CNS disease (Webb and Muir, 2000). Lactate in these situations is one of the best predictive parameters for lesion

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severity and individual mortality (Lagutchik et al., 1996). The statistically significant negative correlation between Blood-L and survival was weak. However, on analysing

survival by establishing a cut-off point for Blood-L at 4 mmol/L, there were statistically

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significant differences. Elevated Blood-L should therefore be considered as a possible

negative prognostic marker, especially in dogs with concentrations higher than 4 mmol/L.

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Isolated hyperlactatemias under 4 mmol/L are likely of no significance.

Few studies have evaluated CSF-L concentrations in healthy dogs but reference values are not clearly established and may also be method and analyser dependent: 1.90

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± 0.61 mmol/L (Pugliese et al., 2005), 1.66 ± 0.22 mmol/L (Taggart and Maxwell, 2011), 1.189 mmol/L (0.416 - 1.850 mmol/L; Galán-Rodríguez et al., 2013), 1.6 ± 0.4 mmol/L

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(Caines et al., 2013), and 2.094 ± 0.374 mmol/L (Musteata et al., 2013). In our study, a

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healthy animal control group could not be used due to ethical reasons. We considered it too invasive to perform a not entirely risk-free technique, such as CSF collection in healthy client-owned dogs. Therefore, our control group was comprised of dogs with a final diagnosis of idiopathic epilepsy but without seizures during the 15 days prior to the diagnosis. It could not be ruled out that the CSF-L values in the IE group (2.78 ± 0.57 mmol/L), which were slightly higher than those published for healthy animals, were a

consequence of the pathology itself. No previous studies have assessed concentrations of CSF-L in epileptic dogs without recent epileptic seizures. The mean CSF-L concentration in the MUO group was 4.10 ± 2.31 mmol/L, significantly higher than in the IE group. Statistically significant differences in the CSF-L values found between the MUO and IE groups may suggest that elevation of this parameter could be associated with the development of MUO. In the MUO group, two dogs experienced seizures in the 15 days prior to the study. The CSF-L concentrations in those dogs were 3.2 and 3.5 mmol/L, very

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close to the mean CFS-L concentration and under our cut off-point (4 mmol/L) which makes it unlikely that these lactate values are the consequence of recent seizure activity,

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although it cannot be ruled out completely.

There was a moderate-strong positive correlation between Blood-L and CSF-L

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concentrations in the MUO group, which was not observed in IE group. In healthy

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individuals, CSF-L concentrations should be independent of Blood-L concentrations. Blood brain barrier (BBB) regulates lactate transport between CSF and blood. Blood-L is found in an ionized form and, in physiological pH ranges, its diffusion through the BBB

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is practically non-existent (Sullivan et al., 2009). Any alterations associated with poor brain oxygenation, high metabolic activity, and/or raised intracranial pressure (which are

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frequent situations in MUO) may increase CSF-L concentrations (Ronquist et al., 1985;

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Petty and Lo, 2002). Our results have evidenced statistically significant differences in CSF-L concentrations between the MUO and IE groups, being higher in the MUO group, which could support the theory that CSF-L might be considered a biomarker of neuronal damage and of an alteration in the BBB in this pathology.

Mean CSF-L (6.47 mmol/L) and Blood-L (7 mmol/L) concentrations were significantly higher at diagnosis in animals submitted to humane euthanasia than in treated groups (Table 2). In addition, Blood-L and CSF-L concentrations in dogs surviving < 7 days (6.27 and 6.84 mmol/L respectively) were significantly higher than in those surviving for > 7 days (2.05 and 3.21 mmol/L). Dogs euthanased at the time of diagnosis or surviving < 7 days presented a more severe and advanced disease which was reflected in their elevated CSF-L and Blood-L concentrations. Therefore, determination

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of these variables could be very valuable for prognosis in MUO.

A total of 67% dogs that had favourable responses to treatment exhibited

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significantly lower Blood-L and CSF-L concentrations than those with unfavourable responses. Thus, Blood-L and CSF-L could be good markers for predicting responses to

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therapy: dogs with lower concentrations of Blood-L and CSF-L will possibly have better

cured after treatment.

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responses. No relationship was found between Blood-L and CSF-L concentrations in dogs

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Statistically significant differences were found when comparing Blood-L and CSF-L concentrations in dogs with favourable and unfavourable outcome. Isolated

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concentrations of lactate should be used as prognostic indicators only for CSF-L and not

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for Blood-L due to the weak-negative correlation between Blood-L and survival time. At higher concentrations of CSF-L, dogs had lower survival rates. Any alteration associated with poor cerebral oxygenation, increased metabolic and immunological activity and/or raised intracranial pressure can lead to a higher CSF-L concentration (Ronquist, Callerud, Niklasson and Friman, 1985; Watson and Scott, 1995). In our study, survival was significantly lower in dogs with CSF-L> 4 mmol/L (Fig. 2). Lobert et al. (2003) found

elevated concentrations of CSF-L in dogs with CNS inflammatory diseases (without differentiating infectious from non-infectious diseases); however, this group did not analyse survival (Lobert et al., 2003). CSF-L > 4mmol/L in our study are associated with lower survival rates and thus could be a useful prognostic indicator. The dogs with CSFL >4 mmol/L showed severe clinical signs and, therefore, a more advanced stage of the disease, which might be explained by the presence of severe neuronal damage. There were only three histopathological confirmations of MUO in this group with evidence of

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severe neuronal damage and these dogs showed lactate concentrations > 4 mmol/L.

The four main limitations of this study are as follows: (1) The CSF analysis as an

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inclusion criterion. This may have generated bias in dogs with increased intracranial pressure, which is usually associated with more severe lesions and poor outcome and

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these dogs were not included in this study because a CSF collection is contraindicated.

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(2) The absence of a reference interval for lactate concentrations in CSF using the Accutrend analyser, but we were not able to establish them in a healthy control group for the reasons described previously. In addition, although our results show statistically

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significant differences in CSF-L concentrations between the MUO and IE groups, bias in samples with very high lactate concentrations cannot be ruled out since Stevenson et

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al., 2007 have described bias for canine blood samples with lactate concentrations >5

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mmol/L using this lactate analyser. (3) Lack of a control group of healthy animals due to ethical reasons involved in subjecting healthy animals to an invasive and risky technique such as a CSF collection. (4) A lack of histopathological confirmation (only obtained in three dogs), which is the main reason why the inclusion criteria (such as performing PCR analysis of infectious diseases in CSF in all dogs) were so strict.

Conclusions Finding simple tests that aid prognosis is a priority in veterinary medicine. The results of this work show, for the first time, that Blood-L and CSF-L concentrations in canine patients with MUO could be used as adjunctive prognostic indicators with a probable cutoff point of 4 mmol/L for both parameters. However, CSF collection is an invasive technique not entirely risk-free and for this reason we recommend including CFS-L measurement in CSF samples collected for other purposes (e.g. cytology, infectious

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diseases). Blood-L and CSF-L concentrations could be considered valuable tools in dogs with MUO, because lactate measurements are simple, fast, inexpensive, reliable, and may

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inform prognosis.

Conflict of interest statement

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None of the authors of this paper have a financial or personal relationship with

the paper.

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Acknowledgments

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other people or organisations that could inappropriately influence or bias the content of

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The authors would like to thank Hill’s Pet Nutrition for the Hill’s Fellowship and the

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Clinical Pathology and Pet Parasite Laboratory, Hospital Clinico Veterinario Complutense for performing the laboratory analysis. The authors also wish to thank Mrs. Paloma Toni for her contribution to the study and Mr. Ricardo Garcia from Universidad Complutense, Madrid Computing Services who conducted the biostatistical analysis.

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Sullivan, L. a., Campbell, V.L., Klopp, L.S., Rao, S., 2009. Blood lactate concentrations in anesthetized dogs with intracranial disease. Journal of Veterinary Internal Medicine. 23, 488–492. Taggart, R., Maxwell, M., 2011. Evaluation of cerebrospinal fluid lactate and glucose levels in acute canine intervertebral disc disease. Proceedings of the American College of Veterinary Internal Medicine Forum, Anahemin, California, 9th-12th June 2010 p. 740. Talarico, L.R., Schatzberg, S.J., 2010. Idiopathic granulomatous and necrotising inflammatory disorders of the canine central nervous system: A review and future perspectives. Jorunal of Small Animal Practice. 51, 138–149.

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Watson, M., Scott, M., 1995. Clinical utility of biochemical analysis of cerebrospinal fluid. Clinical Chemistry. 41, 343–360.

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Webb, A.A., Muir, G.D., 2000. The blood-brain barrier and its role in inflammation. Journal of Veterinary Internal Medicine. 14, 399–411.

Table 1. Blood and cerebrospinal fluid (CSF) analyses in the idiopathic epilepsy (IE) group and meningoencephalitis of unknown origin (MUO) group. IE group n

Mean (range)

Hematocrit (L/L)

11

0.42 (0.35 - 0.55)

27

0.47 (0.36 - 0.61)

Total Solid (g/L)

11

63.5 (45 - 72)

25

67.1 (54 - 80)

White blood cells (x109/L)

11

11.42 (6.10 - 14.90)

26

9.43 (5.80 - 16.92)

Neutrophils (x109/ L)

11

8.85 (4.27 - 11.56)

23

6.65 (3.46 - 11.95)

Band Neutrophils (x109/L)

11

0 (0-0)

23

0.029 (0 – 0.196)

Lactate (mmol/ L)

11

1.74 (0.9 - 3.9)

41

2.77 (0 - 13.20)

Total Proteins (g/L)

11

0.24 (0.14 – 0.3)

40

0.57 (0.17 – 1.74)

Red blood cells/ μL

11

0 (0 - 1)

43

28.1 (0-350)

TNCC (cells/ μL)

11

1 (0 -2 )

43

114.91 (0 - 1720)

Neutrophils (%)

11

NAf

43

7.43 (0 - 61)

Lymphocytes (%)

11

NA

43

15.35 (0 - 86)

Activated lymphocytes a (%)

11

NA

43

22.02 (0 - 91)

11

NA

43

2.62 (0 - 13)

11

NA

43

4.02 (0 - 48)

Activated monocytes c (%)

11

NA

43

28.91 (0 - 81)

Eosinophils (%)

11

NA

43

0.26 (0 - 7)

Lactate (mmol/ L)

11

2.78 (2.10 - 3.8)

45

4.10 (1.80 - 13)

Lymphoblast b (%)

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Mean (range)

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MUO group

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TNCC, total nucleated cell count; CSF, Cerebrospinal fluid; NA, No alteration i.e. no relevant alterations in general cytological evaluation and thus the differential count was not performed a Activated lymphocytes, lymphocyte size up to 1-1.5 times larger than a mature lymphocyte; b Lymphoblast, Lymphocyte 1.5 times larger than a mature lymphocyte c Activated monocyte, Monocyte with vacuoles in its cytoplasm

Table 2 Blood lactate (Blood-L) and cerebrospinal fluid (CSF) lactate (CSF-L) concentrations in the different treatment groups of dogs with meningoencephalitis of unknown origin (MUO). Treatment group

n

A

22 Blood-L 22

2.37 (0.80 – 7.50)

CSF-L

3.25 (1.80 – 6.30)

(Prednisolone + cytosine arabinoside)

2.53 (0.8 – 1.0)

CSF-L

17

4.36 (2 – 13)

Blood-L

3

CSF-L

6

6

7 (3.70 – 13.20)

6.47 (4.20 – 12.30)

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(No treatment)

22

Mean (range; mmol/L)

17 Blood-L 16

(Prednisolone ± cyclosporine) C

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B

Lactate

Table 3 Blood and cerebrospinal fluid (CSF) analysis in the meningoencephalitis of unknown origin group in dogs with survival <7 days and >7 days. n

Survival < 7 days

n

Survival > 7 days

Mean (range) 0.46 (0.42 - 0.53)

22

0.47 (0.36 - 0.61)

Total Solid (g/L)

5

67.2 (54 - 80)

20

67 (58 - 78)

White blood cells (x109/L)

5

10.06 (5.50 - 13.90)

21

9.28 (5.80 - 16.92)

Segmented neutrophils (x109/L)

5

8.05 (3.52 - 11.95)

18

6.27 (3.46 - 10.09)

Band neutrophils (x109/L)

5

0.039 (0 - 0.196)

18

0.026 (0 – 0.186)

Lactate (mmol/L)

5

6.27 (3.70 - 13.20)

34

2.05 (0 - 7.50)

Total Proteins (g/L)

9

0.72 (0.27 – 1.74)

33

0.53 (0.17 – 1.19)

TNCC (cells/ μL)

10

127 (0 - 185)

33

111 (0 - 172)

Neutrophils (%)

10

9.80 (0 - 61)

33

6.71 (0 - 54)

Lymphocytes (%)

10

13.50 (0 - 46)

33

15.91 (0 - 86)

Activated lymphocytes a (%)

10

14.90 (0 - 84)

33

24.18 (0 - 91)

Lymphoblast b (%)

10

2.10 (0 - 7)

33

2.77 (0 - 13)

Monocytes (%)

10

6 (0 - 48)

33

3.42 (0 - 40)

Activated monocytes c (%)

10

23.30 (0 - 75)

33

30.61 (0 - 81)

Eosinophils (%)

10

0 (0 - 0)

33

0.33 (0 - 7)

11

6.84 (4.20 - 13)

34

3.21 (1.8 - 6.30)

Lactate (mmol/L)

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CSF

Hematocrit (L/L)

lP

Blood

Mean (range)

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TNCC, total nucleated cell count; CSF, Cerebrospinal fluid; NA, No alteration i.e. no relevant alterations in general cytological evaluation and thus the differential count was not performed a Activated lymphocytes, lymphocyte size up to 1-1.5 times larger than a mature lymphocyte; b Lymphoblast, Lymphocyte 1.5 times larger than a mature lymphocyte c Activated monocyte, Monocyte with vacuoles in its cytoplasm

Figure legends Fig.1. Kaplan-Meier survival curve for blood lactate (Blood-L) concentrations ≤ 4 and > 4 mmol/L. Dogs with Blood-L > 4 mmol/L had a lower survival time (P = 0.031). Vertical

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lines indicate censored cases.

Fig. 2. Kaplan-Meier survival curve for cerebrospinal fluid lactate (CSF-L) concentrations ≤ 4 and > 4 mmol/L. Dogs with CSF-L > 4 mmol/L had a lower survival time (P = 0.009). Vertical lines indicate censored cases.

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