Serum concentrations of complement C3 and C4 in dogs with idiopathic epilepsy

Abstract

Background

High concentrations of complement factors are presented in serum of animal epilepsy models and human patients with epilepsy.

Objectives

To determine whether complement dysregulation occurs in dogs with idiopathic epilepsy (IE).

Animals

The study included 49 dogs with IE subgrouped into treatment (n = 19), and nontreatment (n = 30), and 29 healthy dogs.

Methods

In this case‐control study, the serum concentrations of the third (C3) and fourth (C4) components of the complement system were measured using a canine‐specific ELISA kit.

Results

Serum C3 and C4 concentrations were significantly higher in dogs with IE (C3, median; 4.901 [IQR; 3.915‐6.673] mg/mL, P < .001; C4, 0.327 [0.134‐0.557] mg/mL, P = .03) than in healthy control dogs (C3, 3.550 [3.075‐4.191] mg/mL; C4, 0.267 [0.131‐0.427] mg/mL). No significant differences were observed in serum C3 and C4 concentrations between dogs in the treatment (C3, median; 4.894 [IQR; 4.192‐5.715] mg/mL; C4, 0.427 [0.143‐0.586] mg/mL) and nontreatment groups (C3, 5.051 [3.702‐7.132] mg/mL; C4, 0.258 [0.130‐0.489] mg/mL). Dogs with a seizure frequency >3 times/month had significantly higher serum C3 (6.461 [4.695‐8.735] mg/mL; P < .01) and C4 (0.451 [0.163‐0.675] mg/mL; P = .01) concentrations than those with a seizure frequency ≤3 times/month (C3, 3.859 [3.464‐5.142] mg/mL; C4, 0.161 [0.100‐0.325] mg/mL).

Conclusions and Clinical Importance

Dysregulation of classical complement pathway was identified in IE dogs. Serum C3 and C4 concentrations could be diagnostic biomarkers for IE in dogs with higher seizure frequency.

Abbreviations

ASM

antiseizure medication

AUC

area under the receiver operator characteristic curve

C3

third component of complement

C4

fourth component of complement

CSF

cerebrospinal‐fluid

IE

idiopathic epilepsy

KBr

potassium bromide

ROC

receiver operator characteristic

1. INTRODUCTION

Idiopathic epilepsy (IE) is the most common type of epilepsy in dogs. ¹ Overall, the prevalence of IE ranges between 0.5% and 5% in the dog population. ² IE is characterized clinically by 2 or more unprovoked epileptic seizures separated by 24 hours with no underlying structural brain lesions or other signs of neurological disease. ³ , ⁴ Approximately 25%‐30% of all epilepsy cases in dogs are drug‐resistant epilepsy. ⁵ Several pathogenic mechanisms are currently being investigated for the treatment of drug‐resistant epilepsy in humans. ⁶

Examination of resected human brain tissue and experimental animal models has elucidated the role of the immune system in epilepsy. ⁷ , ⁸ , ⁹ , ¹⁰ Neuroinflammation is the specific interaction between damaged brain tissue and the innate immune system. ¹¹ It is induced by classical factors such as infections, toxins, and autoimmunity and by triggers that increase neuronal activity such as epileptic seizures. ¹² However, if neuroinflammation is not properly resolved or spreads to remote sites, it can become maladaptive, thereby contributing to the pathogenesis of the disease. Specific inflammatory molecules such as interleukin 1 beta, tumor necrosis factor, cyclooxygenase, complement factors, and high mobility group box 1 are significantly contribute to epileptogenesis in healthy brain and drug resistance in experimental models. ¹³

One of the neuroinflammation pathways is the complement system, a crucial effector of innate immunity and a modulator of adaptive immunity. ¹⁴ The complement system consists of approximately 30 plasma and cell surface proteins that interact with each other to induce a series of inflammatory responses involved in defense against infection. ¹⁵ The third (C3) and fourth (C4) components of complement are the 2 most abundant complement analytes. C3 occupies a cornerstone position where all complement activation pathways converge, while C4 is a key component of the classical complement activation pathway. ¹⁶ Dysregulation of the complement system contributes to the pathogenesis of epilepsy. ¹⁷ Classical pathway component concentrations are elevated in patients with epilepsy relative to those in healthy controls and in patients with uncontrolled compared to controlled epilepsy. ¹⁷ , ¹⁸ , ¹⁹ , ²⁰ , ²¹ These results might contribute to the discovery of potential diagnostic markers and therapeutic targets for epilepsy. Definition of biomarker is a laboratory measurement that reflects the process of disease activity. ²² The discovery of a diagnostic biomarker that can provide criteria for diagnosis and classification, specific to epilepsy, or a monitoring biomarker that could be measured easily in blood, would greatly enhance the treatment of epilepsy in dogs. The severity of seizures is assessed based on seizures that had already occurred, including the total number of seizures, seizure frequency, and seizure free intervals. ²³ There is currently no biomarker for assessing the severity of IE in dogs.

We hypothesized that the complement system might be implicated in the pathogenesis of neuroinflammation in dogs with IE. The aim of this study was to identify whether complement dysregulation occurs in dogs with IE and to investigate whether serum complement concentration can be used as a diagnostic or monitoring biomarker in dogs with IE.

2. MATERIALS AND METHODS

2.1. Animals

Dogs that visited Chungbuk National University Veterinary Teaching Hospital between 2018 and 2022 were recruited for this retrospective case‐control study. Controls were considered clinically healthy if abnormalities were not detected on physical examination, complete blood count, serum biochemical analysis, serum electrolyte analysis, urinalysis, thoracic and abdominal radiography, and abdominal ultrasonography. IE was diagnosed as recurrent seizures of unknown origin accompanied by normal results from various tests, including medical history, physical and neurological examination, laboratory tests, magnetic resonance imaging, cerebrospinal fluid (CSF) and blood analyses, as well as CSF infectious organism antigen titers (canine distemper, Borrelia spp., Ehrlichia canis, Bartonella spp., Blastomyces spp., Cryptococcus spp., Neospora spp., and Toxoplasma spp.). ⁴ Dogs diagnosed with IE and clinically healthy dogs were included in this study. However, dogs with IE that developed any other disease or were diagnosed with IE below the International Veterinary Epilepsy Task Force Tier 2 confidence level were excluded from this study. ⁴

2.2. Grouping and data analysis

Data on characteristics, including sex, age, body weight, seizure type (focal or generalized), duration of epilepsy, time since the last seizure, seizure frequency, treatment period, and epilepsy control status were collected after enrollment. Referring to previous studies that measured inflammatory mediators in human patients with epilepsy, the criteria for dividing the group into 2 subgroups, namely those experiencing 3 or more seizures per month and those experiencing less than 3 seizures per month, were established. ²⁴ The time point for dividing these groups was set as 3 days following seizure onset, in reference to the half‐life of complement factors, which is known to be 72 hours. ²⁵ The seizure information used in the analysis was obtained through data recorded during monthly hospital visits or telephone interviews. To examine the differences in serum complement concentrations according to the antiseizure medication (ASM) treatment, dogs with IE were dichotomized into the nontreatment and the treatment IE groups. For the purpose of this study, well controlled was defined as <1 seizure/month and poorly controlled was defined as ≥1 seizure/month. ²⁶ To examine the differences in serum complement concentrations according to clinical features, the nontreatment IE group was also divided into 2 subgroups depending on the duration of epilepsy, time since last seizure, and seizure frequency, as described previously. ²⁷

2.3. Measurement of C3 and C4

Blood samples (3 mL) of dogs with IE and healthy controls were obtained via jugular venipuncture. Sera were separated from the blood samples by centrifugation at 3500×g at 4°C for 10 minutes within 1 hour of sample collection. Serum samples were stored in 1.5 mL aliquots at −80°C until assayed. Based on human studies of complement biomarkers in epilepsy that have reported high complement concentrations, 2 complement analytes were selected for the present study. ³ , ¹⁶ , ¹⁷ , ²⁰ C3 and C4 concentrations were estimated using a canine‐specific ELISA kit (Dog complement 3 and 4 ELISA kit, Dog Complement 3 ELISA kit and Dog Complement 4 ELISA kit, CUSABIO, Wuhan, China) according to the manufacturer's protocol. Standards were included in each plate, and samples were randomly assigned to eliminate assay bias. Optical density was determined at 450 nm using an automated microplate reader (ELx 808, BioTek Instruments Inc, Winooski, Vermont).

2.4. Statistical analysis

Data were analyzed using commercial statistical software (Prism 9, GraphPad Software Inc., La Jolla, CA, USA) and are expressed as medians and interquartile ranges. A 2‐tailed test was used for analyses. The Shapiro‐Wilk test was performed to determine the distribution (normality) of the data. Differences in sex, age, body weight, and complement concentrations between the healthy controls and dogs with IE were compared using the chi‐square test, Student's t‐test, and Mann‐Whitney U test. Comparisons among healthy controls, nontreatment IE group, and treatment IE group for each analyte were made using the nonparametric Kruskal‐Wallis test, followed by the Dunn's multiple comparison post hoc test. The Student's t‐test and Mann‐Whitney U test were used to compare subgroup differences in the nontreatment IE group. To differentiate between dogs in the treatment, nontreatment, and control groups, the receiver operator characteristics (ROC) area under the curve was used to determine the optimal cutoff values for serum C3 and C4 concentrations. The area under the ROC curve (AUC) was obtained, and the diagnostic accuracy was classified according to the AUC value as follows: sufficient (.6‐.7), good (.7‐.8), very good (.8‐.9), and excellent (.9‐1.0). ²⁸ P < .05 was considered statistically significant. The Spearman or the Pearson tests were used to identify the correlation between the time since the last seizure and C3 or C4, according to normality.

3. RESULTS

3.1. Study cohort

Of the 156 dogs that were seen for seizure, 119 dogs were diagnosed with IE. From these, 70 dogs were excluded because of the presence of concurrent diseases. Finally, this retrospective study included 78 dogs with the consent of their owners. Of these, 29 were healthy controls and 49 were diagnosed with IE. The healthy control dogs consisted of 7 beagles, 4 mixed‐breed, 4 poodles, 2 cocker spaniels, 2 Labrador retrievers, 2 Maltese, 2 Pomeranians, and 1 of each of the following breeds: Cavalier King Charles spaniel, Chihuahua, German shepherd, golden retriever, Siberian husky, and spitz. The dogs with IE comprised 12 Maltese, 10 poodles, 4 bichon frisé, 4 Golden retrievers, 3 Pomeranians, 3 Yorkshire terriers, 2 miniature schnauzers, 2 mixed‐breed dogs, 2 Shih Tzu, 2 spitzes, and 1 of each of the following breeds: border collie, dachshund, Jindo, Labrador retriever, and Pekingese. Dogs with IE and healthy control dogs were comparable without any significant differences in sex, age, and body weight. Among the dogs with IE, 43 had generalized seizure type and 6 had focal seizure type. The dogs with IE included the nontreatment IE group (30 dogs) and the treatment IE group (19 dogs). The treatment IE group was treated with ASM, phenobarbital or potassium bromide (KBr), only. The demographics and clinical features of all the groups are shown in Table 1.

TABLE 1.

Demographic and clinical data of the study cohort.

	Idiopathic epilepsy (n = 49)	Healthy control (n = 29)	P‐value
Sex (M/F)	31/18 (63.3/36.7%)	14/15 (48.3/51.7%)	.20
Neutered (M/F)	24/14 (49.0/28.6%)	10/6 (34.5/20.7%)
Age (months)	57 (36‐96)	61 (46‐85)	.66
Body weight (kg)	5.78 (3.95‐8.53)	9.30 (4.98‐14.55)	.21
Age at onset (months)	43 (27‐61)	–
Seizure type
Generalized	43 (87.8%)	–
Focal	6 (12.2%)	–
Administration of ASM
Phenobarbital	17 (34.7%)	–
KBr	2 (4.1%)	–
None	30 (61.2%)	–

Note: Age, body weight, and age at onset are expressed as medians and interquartile ranges. The values of sex, seizure type, and administration of ASM were expressed as the number of dogs (percentage of total instances). Age at onset is the age at which the first seizure occurred.

Abbreviations: ASM, antiseizure medication; F, female; KBr, potassium bromide; M, male.

3.2. Comparison of the serum C3 concentrations between healthy and dogs with IE

The serum C3 concentrations of dogs with IE (median, 4.901 [interquartile range, 3.915‐6.673] mg/mL) were significantly higher than those of healthy control dogs (3.550 [3.075‐4.191] mg/mL; P < .001; Figure 1A). Multiple comparisons were performed among the healthy controls, nontreatment IE (5.051 [3.702‐7.132] mg/mL), and treatment IE (4.894 [4.192‐5.715] mg/mL) groups. In comparison to healthy controls, serum C3 concentrations were statistically significantly increased in the nontreatment (P < .001) and treatment (P < .01) groups but did not statistically significantly differ between the nontreatment and treatment IE groups (Figure 1B).

FIGURE 1.

(A) Serum complement 3 (C3) concentrations of healthy control dogs compared to those of dogs with idiopathic epilepsy (IE; Mann–Whitney U test). (B) serum C3 concentrations of healthy controls compared to those of nontreatment and treatment IE group (Kruskal‐Wallis test followed by post hoc Dunn's multiple comparison test). The horizontal lines indicate the medians and interquartile ranges. The asterisk indicates statistically significant difference (***P < .001).

3.3. Comparison of the serum C4 concentrations between healthy and IE dogs

The serum C4 concentrations of dogs with IE (0.327 [0.134‐0.557] mg/mL) were statistically significantly higher than those of healthy control dogs (0.267 [0.131‐0.427] mg/mL; P = .03; Figure 2A). Multiple comparisons were performed among the healthy controls, nontreatment (0.258 [0.130‐0.489] mg/mL), and treatment IE (0.427 [0.143‐0.586] mg/mL) groups. No statistically significant differences were observed between the groups with respect to serum C4 concentrations (P = .19; Figure 2B).

FIGURE 2.

(A) Serum complement 4 concentrations of healthy control dogs compared to those of dogs with idiopathic epilepsy (IE; Mann‐Whitney U test). (B) serum complement 4 concentrations of healthy controls compared to those of nontreatment and treatment IE group (Kruskal‐Wallis test followed by post hoc Dunn's multiple comparison test). The horizontal lines indicate the medians and interquartile ranges. The asterisk indicates a statistically significant difference (*P < .05).

3.4. Serum C3 and C4 concentrations of dogs with IE according to the epilepsy treatment

The time since the last seizure was statistically significantly shorter in the nontreatment IE group (2 [0‐8.25] days) than in the treatment IE group (60 [1‐240] days; P = .04), but there were no statistically significant differences in serum C3 and C4 concentrations between the groups. The treatment IE group underwent ASM treatment for 46‐1898 days (median, 366 days) and was classified into the well‐controlled (<1 seizure/month; 10 dogs) and poorly controlled (≥1 seizure/month; 9 dogs) groups, according to the treatment response. There were no statistically significant differences in serum C3 concentrations between dogs in the well‐controlled group (median, 4.733 [3.884‐6.066] mg/mL) and those in the poorly controlled group (4.901 [4.328‐6.300] mg/mL; Figure 3A). Similar results were obtained for serum C4 concentrations between dogs in the well‐controlled group (0.232 [0.110‐0.511] mg/mL) and those in the poorly controlled group (0.543 [0.397‐0.684] mg/mL; Figure 3B).

FIGURE 3.

(A) Serum complement 3 concentrations in dogs with well‐controlled compared to those in dogs with poorly controlled (Mann‐Whitney U test). (B), serum complement 4 concentrations in dogs with well‐controlled compared to those in dogs with poorly controlled (Mann‐Whitney U test). The horizontal lines indicate the medians and interquartile ranges.

3.5. Subgroup comparison of serum C3 and C4 concentrations in the nontreatment IE group

No differences in serum C3 and C4 concentrations were observed between the groups based on the duration of epilepsy or time since the last seizure (Table 2). Dogs with a seizure frequency >3 times/month had significantly higher serum C3 (P < .01) and C4 (P = .01) concentrations than those with a seizure frequency ≤3 times/month. No statistically significant correlation was identified between C3 (P = .38, r = −.16) and C4 (P = .74, r = −.06) concentrations and time since last seizure.

TABLE 2.

Subgroup comparisons of serum C3 and C4 concentrations in the nontreatment IE group.

	Nontreatment IE group (n = 30)
	Number (cases)	C3 (mg/mL)	C4 (mg/mL)
Duration of epilepsy
≤6 months	14	4.899 (3.769‐7.949)	0.420 (0.176‐0.524)
>6 months	16	5.142 (3.689‐6.321)	0.163 (0.095‐0.477)
P value		.76	.42
Time since last seizure
≤3 days	19	5.209 (4.422‐7.070)	0.327 (0.136‐0.486)
>3 days	11	3.971 (3.540‐7.317)	0.183 (0.110‐0.629)
P value		.63	.89
Seizure frequency
≤3 times/month	13	3.859 (3.464–5.142)	0.161 (0.100–0.325)
>3 times/month	17	6.461 (4.695‐8.735)	0.451 (0.163–0.675)
P value		<.01	.01

Note: Data represented as median (interquartile range).

Abbreviations: C3, third component of complement; C4, fourth component of complement; IE, idiopathic epilepsy.

3.6. ROC curve of serum C3 and C4 concentration in dogs with IE

The ROC curve illustrates the sensitivity and specificity of serum C3 and C4 concentrations in differentiating healthy dogs from those with IE or untreated IE (Figure 4). The AUCs of serum C3 concentrations were: (1) dogs with IE and healthy controls, 0.81 (95% confidence interval [CI]: 0.72‐0.90) and (2) nontreatment IE group and healthy controls, 0.80 (95% CI: 0.69‐0.91). The corresponding optimal cutoffs (sensitivity and specificity) for serum C3 concentration were: (1) dogs with IE and healthy controls, 4.461 mg/mL (67.4% [95% CI: 53.4%‐78.8%] and 89.7% [95% CI: 73.6%‐96.4%]), (2) nontreatment IE group and healthy controls, 4.576 mg/mL (63.3% [95% CI: 45.5%‐78.1%] and 93.1% [95% CI: 78.0%‐98.8%]), respectively. The AUC of serum C4 concentrations for dogs with IE and healthy controls was 0.60 (95% CI: 0.48‐0.73). The corresponding optimal cutoffs (sensitivity and specificity) for the serum C4 concentration were: dogs with IE and healthy controls, 0.455 mg/mL (36.7% [95% CI: 22.9%‐48.7%] and 89.7% [95% CI: 73.6%‐96.4%]). There was no significant difference in the AUC of serum C4 concentrations between nontreatment IE group and healthy controls.

FIGURE 4.

ROC curve analysis of predicting the serum C3 concentration between (A) dogs with IE and healthy controls, (B) nontreatment IE group and healthy controls, and the serum C4 concentration between (C) dogs with IE and healthy controls. The dotted diagonal line represents the area under the curve that is 50%. The AUCs of the ROC curve line marked by rectangle symbols were 0.81 (95% CI: 0.72‐0.90; A), 0.80 (95% CI: 0.69‐0.91; B), and 0.60 (95% CI: 0.48‐0.73; C), respectively. The point of intersection in all ROC curves represents the optimal cutoff value (sensitivity and specificity) of 4.461 mg/mL (67.4% [95% CI: 53.4%‐78.8%] and 89.7% [95% CI: 73.6%‐96.4%]; A), 4.576 mg/mL (63.3% [95% CI: 45.5%‐78.1%] and 93.1% [95% CI: 78.0%‐98.8%]; B), and 0.455 mg/mL (36.7% [95% CI: 22.9%‐48.7%] and 89.7% [95% CI: 73.6%‐96.4%]; C), respectively. AUC, area under the receiver operating characteristic curve; C3, third component of complement; C4, fourth component of complement; CI, confidence interval; IE, idiopathic epilepsy; ROC, receiver operating characteristic.

4. DISCUSSION

In the present study, serum C3 and C4 concentrations were statistically significantly higher in dogs with IE than in the healthy control dogs. No difference was noted in serum C3 and C4 concentrations between the nontreatment and treatment IE groups. In addition, the serum C3 and C4 concentrations of dogs with a seizure frequency >3 times/month were significantly higher than those with a seizure frequency ≤3 times/month. These results suggest that serum C3 and C4 concentrations might be biomarkers for IE diagnosis or might be associated with pathogenesis in dogs with IE.

The pathogenesis of IE is yet to be elucidated. ²⁹ Neuroinflammation has recently been associated with epileptogenesis. ²⁹ , ³⁰ , ³¹ However, although there is evidence of this relationship in dogs with IE, ²⁶ no studies have evaluated the involvement of the complement cascade. The components of the complement cascade in humans are known to mediate inflammation in the nervous system by increasing vascular permeability, inducing chemokines and adhesion molecules, recruiting immune cells, activating glial cells, and enhancing the production of proinflammatory cytokines. ³² Since the expression of classical complement pathway proteins is increased in regions with neuronal cell loss, as well as in reactive astrocytes and microglia or macrophages, ¹⁸ activation of the classical complement pathway might contribute to the initiation or maintenance of neuroinflammation in dogs with IE. The results of the present study were consistent with those of previous reports on human patients with temporal lobe epilepsy and idiopathic epilepsy, ¹⁸ , ³³ showing higher complement concentrations in dogs with IE than in healthy controls. Therefore, the present study shows that dysregulation of the classical complement pathway coupled with increased inflammatory drivers might be present in dogs with IE.

Further analysis showed that the serum C3 concentrations in the nontreatment and treatment IE groups were significantly higher compared to healthy control dogs, but serum C4 concentrations were not higher. The C3 AUCs for differentiating between dogs with IE and healthy control dogs showed good diagnostic accuracy, while C4 AUC had sufficient diagnostic accuracy. ²⁸ The reason for these differences between C3 and C4 could be because of a larger interindividual variability in serum C4 concentrations. The C4 copy number variation is 1 of the major determinants of plasma or serum C4 concentrations in humans. ³⁴ As in humans, the C4 gene is the most polymorphic of all genes encoding complement components in dogs. ³⁵ Therefore, serum C3 concentration could be a useful tool for diagnosing IE in dogs, whereas serum C4 concentrations should be interpreted carefully. If the complement pathway is specifically expressed only in IE in dogs, it could potentially serve as a diagnostic biomarker for IE in dogs. Furthermore, if the complement pathway is only involved in IE in dogs, it could represent a novel therapeutic target.

To determine if there is a relationship between serum C3 and C4 concentrations and ASM treatment responses, comparisons of serum C3 and C4 concentrations with ASM treatment and epilepsy control status were performed. Although the time since last seizure was significantly prolonged in the treatment IE group compared to that in the nontreatment IE group, no differences in serum C3 and C4 concentrations was observed between the 2 groups. In addition, no significant differences between serum C3 and C4 concentrations according to epilepsy control status were identified. In humans complement concentrations decline as epilepsy treatment progressed and administration of sodium valproate, clobazam, and perampanel is associated with changes in the concentrations of serum complement analytes. ¹⁷ , ³³ The reason for the difference in results between previous studies and the present study is presumed to be the difference in the commonly used ASM between humans and dogs. The commonly used ASMs, including valproate, carbamazepine, and phenytoin, in human patients with epilepsy have direct effects on the immune system. Although the mechanism is not clear, ASMs can affect both humoral and cellular immunity, modifying the synthesis and expression of molecules such as cytokines. ³⁶ No study has assessed the effects of phenobarbital and KBr, the most common ASMs for dogs, on the immune system. Therefore, the difference in the routinely prescribed ASMs might explain the differences in the effects of ASMs on serum complement concentrations in dogs and humans with epilepsy.

In the present study, factors including duration of epilepsy, time since last seizure, and seizure frequency were investigated in the nontreatment IE group; the only significant result was found with regard to seizure frequency. Serum C3 and C4 concentrations in dogs with IE showing seizure frequency >3 times/month were significantly higher than those in dogs with seizure frequency ≤3 times/month. Seizure type and frequency are not correlated with serum complement concentrations in humans. ¹⁷ A limitation of this study was the heterogeneity of epilepsy samples with regard to drug treatment. However, the effect of ASM was excluded in the present study by performing comparisons within the nontreatment group. In addition, hippocampal levels of inactivated complement component‐3b, another classical complement pathway component correlate with the frequency of spontaneous seizures in experimental epilepsy models. ³⁵ Thus, serum C3 and C4 concentrations might be associated with seizure frequency in dogs with IE. However, it is uncertain whether increased serum C3 and C4 concentrations are the cause or result of seizures. It is possible that the increase in complement concentrations occurred because of recent seizures. ³⁷ In the present study, there was no correlation between C3 and C4 concentrations and time since the last seizure. It was surmised that elevated serum C3 and C4 concentrations were not a direct consequence of the recent seizures. These results suggest that dysregulation of the complement system might be a cause of poor seizure control.

This study has limitations. First, this study is not a hypothesis driven case‐control study, but a nonhypothesis driven orientation study. A power calculation could not be performed because of the no previous quantitative data about serum concentrations of C3 and C4 in dogs with IE. Also, the small number of dogs included might have contributed to the false negative findings (type 2 error). However, positive findings through the post hoc analysis such as the elevation of serum complement concentrations in IE in dogs could serve as a basis for future research and could be used to perform a sample size calculation for a larger cohort study. Second, complement analytes were not measured in the CSF. This study solely focused on circulating plasma biomarkers as potential predictors of what is occurring within the central nervous system. However, the blood‐brain barrier in dogs and humans with epilepsy is dysfunctional and leaky, ³⁸ , ³⁹ and serum complement are altered in rats with chronic epilepsy. ¹⁸ Thus, the use of serum complement concentrations that are easily accessible and measurable has been well established. Third, complement analytes other than C3 and C4 were not investigated in the present study. Numerous systemic factors might influence the serum concentrations of complement components and inflammatory proteins, especially when a single determination is performed.

CONFLICT OF INTEREST DECLARATION

Authors declare no conflict of interest.

OFF‐LABEL ANTIMICROBIAL DECLARATION

Authors declare no off‐label use of antimicrobials.

INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE (IACUC) OR OTHER APPROVAL DECLARATION

Approved by the Chungbuk National University Animal Care Committee and was carried out according to the Guide for Care and Use of Animals (Chungbuk National University Animal Care Committee, CBNUA‐2005‐22‐02).

HUMAN ETHICS APPROVAL DECLARATION

Authors declare human ethics approval was not needed for this study.

Kang S, Koo Y, Yun T, et al. Serum concentrations of complement C3 and C4 in dogs with idiopathic epilepsy. J Vet Intern Med. 2024;38(2):1074‐1082. doi: 10.1111/jvim.17008