Acute cerebral ischaemia: Relationship between serum and cerebrospinal fluid orexin-A concentration and infarct volume

Abstract

Objective

Orexins are hypothalamic neuropeptides that are involved in feeding, neuroendocrine regulation, sleep-wakefulness and sleep disorders (such as narcolepsy). This study investigated the relationship between serum and cerebrospinal fluid (CSF) orexin-A concentrations and infarct volume, in patients with ischaemic stroke.

Methods

Serum and CSF concentrations of orexin-A were determined 48–72 h after the onset of ischaemic stroke in patients, then compared with those of healthy control subjects of comparable age. Infarct volumes were measured using computerized tomography, 48–72 h after hospitalization.

Results

Mean serum and CSF orexin-A concentrations were significantly lower among ischaemic stroke patients (n = 29) compared with control subjects (n = 13). There was a significant inverse correlation between infarct volumes and CSF orexin-A concentrations in patients with ischaemic stroke.

Conclusion

These data show that serum and CSF orexin-A concentrations decrease after cerebral ischaemia and may play a role in the development of brain injury. The orexin-A concentration in the CSF might be a useful biomarker for the assessment of progression of brain tissue damage during the early stages of ischaemic stroke.

Introduction

Stroke is a life-threatening condition and one of the leading causes of morbidity and mortality worldwide. ¹ This neurological deficit appears over a few hours, persists for >24 h, and is presumed to be due to an impairment of the blood supply to one region of the brain. ¹

Two novel neuropeptides, orexin-A and orexin-B, have been isolated from the mammalian hypothalamus. ² Orexin-A is identical in humans, mice, rats and sheep, whereas human orexin-B differs from murine orexin-B by two amino acids. ³ The orexins bind to two G-protein-coupled receptors: orexin-receptor-1 and orexin-receptor-2. ⁴ Neurons containing orexin are found projecting diffusely into numerous brain regions including the cortex, thalamus, brainstem and spinal cord; ⁵ this pattern of efferent fibre distribution suggests that the orexinergic system may play a role in the regulation of multiple brain functions.^6,7 The ventral tegmental area and nucleus accumbens receive extensive input from the lateral hypothalamus area, including from neurons containing orexin-A and orexin-B. ⁸ In addition, electrophysiological mechanisms have been proposed through which orexins can activate ventral tegmental dopamine neurons directly. ⁹ Orexins are responsible for neuroendocrine regulation, feeding, the control of normal sleep and wakefulness, and sleep disorders.^10,11 It has been shown that intracerebroventricular injections of orexins upregulate c-fos in many different cerebral nuclei, except those in the lateral magnocellular division. ¹² Orexin-A crosses the blood–brain barrier by diffusion. ¹³ A deficit in orexin may be responsible for excessive somnolence, such as secondary narcolepsy, through direct or indirect damage to the posterior hypothalamus and its connections. ¹⁴

Although some studies that have investigated orexin-A concentrations in patients with haemorrhagic stroke,^15,16 to the authors’ knowledge, none have been published on serum and cerebrospinal fluid (CSF) concentrations of orexin-A in patients with ischaemic stroke. The present study investigated the ability of serum and CSF orexin-A concentrations to predict ischaemic stroke in patients with acute ischaemic infarct, by determining whether there was a correlation between serum and CSF orexin-A concentrations and infarct volume.

Patients and methods

Study population

Consecutive patients who presented with acute ischaemic stroke at the Neurology Clinic, Ataturk University Faculty of Medicine, Erzurum, Turkey, between May 2005 and December 2005, were recruited to this cross-sectional study. Patients with ischaemic stroke were selected for inclusion based on a computerized tomography (CT) scanning report, and were matched with healthy controls by age, sex and risk factors for stroke (such as hypertension, diabetes mellitus and hyperlipidaemia). A detailed history of vascular risk factors was obtained for each patient. Stroke severity was assessed using the National Institutes of Health Stroke Scale (NIHSS), which measures stroke severity on a 42-point scale. ¹⁷ All patients were examined within 24 h of admission by investigators who were certified in the application of the NIHSS. Inclusion criteria for patients with ischaemic stroke were a focal neurologic deficit and evidence of an infarct on a CT scan. Patients with transient ischaemic attack and reversible ischaemic neurological deficits were excluded.

Control serum and CSF samples were obtained from age-matched controls who were undergoing lumbar puncture for the administration of intrathecal anaesthetic drugs during orthopedic surgery. None of these subjects had evidence of systemic or central nervous system diseases such as ischaemic or haemorrhagic stroke.

The Institutional Ethics Committee of the Faculty of Medicine, Ataturk University approved the study, and all of the participants provided written informed consent.

Clinical and laboratory assessments

To identify the potential mechanism of cerebral infarction, the following analyses were performed in each patient: complete blood count; leucocyte differential; blood biochemistry (including measurement of blood glucose, glycosylated haemoglobin, triglycerides, total cholesterol, high-density lipoprotein cholesterol and low-density lipoprotein cholesterol levels); systolic and diastolic blood pressure measurement; CSF analysis. Electrocardiography, chest radiography and carotid ultrasonography examinations were also performed, to distinguish between cases of embolic or thrombotic infarct.

Blood/CSF samples and orexin-A measurements

Serum and CSF samples were collected within 48–72 h of admission to hospital. In each participant, ∼2 ml of blood was collected from the antecubital vein into vacuum tubes. After sampling, tubes were immediately centrifuged at 1500  g for 10 min at room temperature, to separate the serum. Aliquots of serum were stored at −80 °C, and routine haematology and biochemistry analyses were undertaken within a few hours after blood sampling. In addition, ∼2 ml of CSF was taken and stored immediately at −80 °C until analysis. Orexin-A was measured, in a manner blinded to diagnosis, by direct radioimmunoassay of 1 ml of serum and 1 ml of CSF, using commercially available kits (Phoenix Pharmaceuticals, Burlingame, CA, USA; detection limit 40 pg/ml; intra-assay variation 5%). Samples with undetectable concentrations (values <40 pg/ml) were operationally plotted at 0 pg/ml.

A routine CT scan of the brain was undertaken to quantify the infarct volume in each stroke patient at baseline, and at 48–72 h after admission to hospital.

Statistical analyses

All statistical analyses were performed using the SPSS® statistical package, version 12.0 (SPSS Inc., Chicago, IL, USA) for Windows®. Statistical significance for intergroup differences was assessed by Pearson’s χ²-test for categorical variables and by Student’s t-test or Mann–Whitney U-test for continuous variables. To study the correlation between quantitative variables, Pearson’s correlation coefficient or Spearman’s rank correlation coefficient tests were used. A P-value < 0.05 was considered statistically significant.

Results

A total of 29 patients with ischaemic stroke and 13 control subjects were included in the study. Their demographic and clinical characteristics are presented in Table 1. There were no significant differences observed between the two groups except for the mean serum and CSF orexin-A concentrations, which were significantly lower in patients with ischaemic stroke compared with control subjects (P < 0.05 for both). The mean ± SD NIHSS scores for all patients with ischaemic stroke were 12.3 ± 7.9. In patients with ischaemic stroke, the CSF orexin-A concentration was inversely correlated with the infarct volume (r = −0.359, P < 0.05; Figure 1). There was no significant correlation between the serum orexin-A concentration and the infarct volume.

Figure 1.

Correlation between cerebrospinal fluid orexin-A concentration (x-axis, pg/ml) and cerebral infarct volume (y-axis, measured in cm³; r = −0.359, P < 0.05) in patients with ischaemic stroke (n = 29).

Table 1.

Demographic and clinical characteristics of patients with acute ischaemic stroke and healthy control subjects, participating in a study investigating the relationship between serum and cerebrospinal fluid orexin-A concentrations and infarct volum.

Characteristic	Control group n = 13	Stroke group n = 29	Statistical significance a
Age, years, range	61.3 ± 7.9, 48 – 68	68.9 ± 9.4, 58–79	NS
Sex, male/female	6/7	13/16	NS
Systolic blood pressure, mmHg	115.0 ± 25.0	155.0 ± 30.0	NS
Diastolic blood pressure, mmHg	75.0 ± 9.8	87.0 ± 9.2	NS
Blood glucose, mg/dl	81.0 ± 11.0	121.0 ± 14.0	NS
Glycosylated haemoglobin, %	4.5 ± 1.9	4.9 ± 1.9	NS
Triglycerides, mg/dl	169.0 ± 48.0	214.0 ± 57.0	NS
Total cholesterol, mg/dl	191.0 ± 39.0	227.0 ± 41.0	NS
HDL-C, mg/dl	47.0 ± 11.0	41.0 ± 9.0	NS
LDL-C, mg/dl	117.0 ± 21.0	139.0 ± 31.0	NS
Serum orexin-A, pg/dl	39.2 ± 12.8	32.2 ± 9.6	P < 0.05
CSF orexin-A, pg/dl	21.4 ± 5.3	16.2 ± 6.4	P < 0.05

Data presented as mean ± SD.

a

Pearson’s χ²-test for categorical variables; Student’s t-test or Mann–Whitney U-test for continuous variables.

HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; CSF, cerebrospinal fluid; NS, not statistically significant (P ≥ 0.05).

Discussion

The present study demonstrated a significant decrease of orexin-A concentrations in the CSF and serum within 48–72 h of stroke onset in patients with ischaemic stroke, compared with control subjects. The most important risk factors for stroke and cardiovascular disease are diabetes, hypertension, hyperlipidaemia and smoking.^18,19 Examining the relationship between traditional risk factors for cardiovascular disease and stroke might clarify the associations between CSF orexin-A concentrations and cerebrovascular infarction. Yuan et al. ²⁰ showed that orexin-A treatment can induce prominent neuroprotective effects on transient cerebral ischaemia in rats by improving neurological function and decreasing infarct size. In the authors’ opinion, serum orexin-A is not a useful indicator for the prediction of symptomatic brain ischaemic infarction, but lower orexin-A concentrations in the CSF may be related to ischaemic injury. The present study suggested that in humans, orexin-A may alter the pathological mechanisms involved in brain ischaemia and indicate that it may have a neuroprotective effect. Thus, measurement of CSF orexin-A concentrations may be useful as a surrogate marker for the detection of ischaemic stroke.

There was a significant inverse correlation between the CSF orexin-A concentration and the brain infarct volume. The authors believe that this is the first study to provide evidence of decreased serum and CSF orexin-A concentrations in patients within 72 h of acute brain injury caused by ischaemic stroke. The findings seem important in light of a study that investigated long-term outcomes in ischaemic infarct, which demonstrated that >75% of patients reported excessive fatigue or daytime sleepiness that persisted for a long period (months to years) after the event. ²¹ The exact mechanism underlying the symptoms of fatigue and daytime sleepiness remain unknown, however.^22,23 Although the aetiology may be quite complex, an abnormality in the orexin system could contribute to this phenomenon. It is, however, not clear how ischaemic stroke affects hypothalamic function.

A lower orexin-A concentration in the CSF was associated with a larger infarct volume, which possibly reflected the intensity of the initial ischaemic injury. Further study into the underlying pathophysiology of this relationship is needed. The limited number of patients included in our study and the absence of long-term results appear to be possible limitations of our research. Consequently, more detailed research, involving larger numbers of participants, is required.

In conclusion, the present study demonstrated an inverse relationship between CSF orexin-A concentration and infarct volume, in patients with cerebral ischaemia, which might provide new insights into the pathogenesis of ischaemic stroke. In the authors’ opinion, the orexin-A concentration in the CSF might be a useful biomarker for the assessment of progression of brain tissue damage during ischaemic stroke.