Depressive symptoms and neurotrophin levels in ostomy patients

Objective: The aim of the present study was to investigate the depressive symptoms and changes in neurotrophins (BDNF, NGF, NT-3), and cortisol levels in serum of peripheral blood from ostomy patients compared to healthy control group. Methods: We evaluated ostomy (n = 29) and healthy control (n = 30) patients. The neurotrophin (BDNF, NGF, NT-3), and cortisol levels were assessed by ELISA in serum of peripheral blood. Depressive symptoms were defined based on the Hamilton Depression Rating Scale (HDRS), and major depression disorder was based on clinical interviews and was confirmed with the Structured Clinical Interview for DSM-IV Axis I Disorders (SCID-I). Results: The results showed a significant decrease in BDNF levels and, a significant increase in NT-3 levels in serum of peripheral blood from ostomy patients when compared to healthy controls. The levels of NGF and cortisol showed no significant differences between groups. The depressive symptom evaluations by HDRS demonstrated a significant increase in ostomy patients when compared to healthy controls. The major depression disorder diagnosis by SCID-I showed no significant difference between groups. Conclusion: Our results suggest ostomy triggers significant depressive symptoms and alterations in neurotrophins levels in serum of peripheral blood samples collected from these patients.


INTRODUCTION
Ostomy is a surgically created opening between an internal organ and the body surface, often created as an intestinal 1 or colon 2 surgical part in cases where the body has no natural possibility of releasing intestinal waste. Also, it is necessary to treat complicated abdominal problems or to allow the healing of surgical site 1 . The ostomy is classified into some types, based on patients' conditions, such as Colostomy, Temporary Colostomy, Permanent Colostomy, Sigmoid or Ascending Colostomy, Transverse Colostomy, Loop Colostomy, Ileostomy, Ileoanal Reservoir (J-Pouch), Continent Ileostomy (Kock Pouch), Urostomy, Continent Urostomy, Indiana Pouch, Neobladder Orthotopic 3 . Each one specifically used, while a urostomy can be formed for a bladder cancer or interstitial cystitis 2 , the ileostomy or colostomy is indicated for recovering more sick or operated parts induced by intestinal inflammatory or malignant diseases, such as: colorectal cancer, diverticular disease, intestinal infarction, severe trauma and also in complex anal complications 4 .
The main ostomy patient issues after discharge are: peristomal skin irritation, pouch leakage, odor, reduction of previously appreciated activities and depression or anxiety [5][6][7] . In clinical trials, it is possible to see that stoma use significantly reduces the quality of life (QOL), and presenting depressive symptoms after the stoma surgery according to ostomy site 6,8 . The overall prevalence of depression was 24% among rectal cancer survivors with the highest prevalence among those with a previous permanent ostomy (31%) 7 .
Ostomy patients can have negative attitudes toward themselves and should have postoperative psychological support 9 . Thus needing a better understanding of these ostomy-related complications to improve coping strategies; in addition, abnormal psychological states may have consequences for self-care and facilitate the several comorbidities occurrence in these patients 6 . Depressive mood was accompanied by several physiological changes 10 . In the present study, we focused on the possible changes in cortisol and neurotrophins levels, and this biomarker relation with depression and ostomy 11,12 . This marker relationship in ostomy patients is still poorly described.
The hypothalamic-pituitary-adrenal axis (HPA) is the central modulator of mammalian neuroendocrine stress response systems, and as such has been important in psychiatric disorders studies [13][14][15][16] . In stress situations, the adrenal gland increases the cortisol production. The increased cortisol release, due to long-term stressful situation exposure, seems to play an important role in the depressive symptoms neurobiology 17 .
Neurotrophins are growth factors that promote the neuronal survival, development and function, acting in the apoptosis prevention, among the neurotrophins are brainderived neurotrophic factor (BDNF), nerve growth factor (NGF) and neurotrophin-3 (NT-3) 18 . The main neurotrophin described in the depression neurobiology is BDNF, either by controlling neuronal number or in the dendritic growth 19 . Studies suggest that BDNF deregulation occurs under chronic-stress conditions and depression 20 . In previous studies, with lower peripheral BDNF levels in depressed patients, BDNF has been considered an important biomarker for Major Depression Disorder (MDD), and antidepressants would be able to reverse this decrease [21][22][23] .
In this context, the present study was aimed at investigating the neurotrophins (BDNF, NGF, NT-3) and cortisol levels changes in peripheral blood taken from ostomy patients when compared to those collected from subjects in a healthy control group.

Patients
The present study was approved by the Ethics Committee of Nursing Clinic of the Unesc, located in the city of Criciúma, Brazil. Patients were only included in the study after signing the Subject Information and Consent Form.

Study design
We conducted a case-control study. A total of 29 consecutives patients over 18 years old with ostomy were selected as cases. The control group was 30 healthy patients, from of the same Nursing Clinic.
Structured questionnaires were administered by the same manner in cases and controls. Information about demographic factors and lifestyle was obtained. Psychiatric diagnoses for major depressive disorder were used in the Structured Clinical Interview for the DSM-IV Axis I Disorders (SCID-I) chapter mood disorders. Depressive symptoms were assessed using the Hamilton Depression Rating Scale (HDRS) patients who scored more than eight were considered cutoff score for presence of mood and depressive symptoms.

Sample collection
From each subject, 10 ml of peripheral blood were drawn by venipuncture into a vacuum tube. Immediately, the blood was centrifuged at 3,000x g for 10 min, and the serum was stored at -80 ºC for subsequent assay.

BDNF, NGF, and NT-3 level measurement
Biochemical analyses were in serum of peripheral from ostomy patients and healthy control patients. For analyzing the neurotrophic factors, the serum were diluted in phosphatebuffered solution (PBS) with 1 mMphenylmethylsulfonyl fluoride (PMSF) and 1 mM ethylene glycolbis (2-aminoethyl ether)-N,N,N'N'-tetraacetic acid (EGTA). The diluted blood serum were centrifuged at 10,000x g for 20 min, and the supernatants were collected for neurotrophic factor level quantification. The BDNF, NGF, and NT-3 levels in serum were measured by sandwich enzyme-linked immunosorbent assay using commercial ELISA kits (96-well plate) according to manufacturer's instructions. NGF and BDNF levels were assessed with a kit from Chemicon (USA) (NGF catalog number: ABIN365470; BDNF catalog number: KE00096), and NT-3 levels were assessed with a kit from Biosensis (USA) (NT-3 catalog number: BEK-2221-2P). Total protein was measured by Lowry et al. 24 method using bovine serum albumin as a standard.

Measurement of cortisol levels
For the quantitative measurement of Cortisol in serum of peripheral blood was diluted in phosphate-buffered solution (PBS) with 1 mMphenylmethylsulfonyl fluoride (PMSF) and 1 mM ethylene glycolbis (2-aminoethyl ether)-N,N,N'N'tetraacetic acid (EGTA). The dilution were centrifuged at 10,000x g for 20 min, and the supernatants were collected for quantification of the cortisol levels. The Cortisol levels in serum were measured by sandwich enzyme-linked immunosorbent assay using commercial kits according to the manufacturer's instructions from Parameter TM (Catalog Number KGE008B). Total protein was measured by Lowry et al. 24 Table 1 shows ostomy patient characteristics when compared to healthy control, the data indicated groups were homogeneous regarding age, gender, smoking, ethnicity, alcoholic, other diseases, and menopause and hormone replacement therapy in the women. The variables that had a significant difference between the groups were: marital status, occupation, and religion. However, these variables do not interfere with the biochemical parameters. Regarding the depressive symptoms evaluations by HDRS and the depressive disorder diagnosis by SCID-I, only HDRS had a significant difference between the groups. It was found an increase in the HDRS (p = 0.027) score was observed in ostomy patients when compared to healthy controls. Table 2 describes the ostomy patient characteristics. The ostomy time median was 26 months and interquartile range (IQL) 11.0-62.5. The most predominant type of ostomy was colostomy (n = 23, 79.3%). 22 patients had neoplasia (75.8%) as the leading cause of ostomy, followed by diverticulitis (n = 3, 10.3%). Variable reversal attempts indicated 25 patients (86.2%) not tried reversing the ostomy. 22 patients (75.9%) reported to used one piece. 17 patients (58.6.7%) reports have no skin complication. The most predominant of patients do not have complications due to ostomy (n = 13, 42.9%). Table 3 describes the biochemistry parameters dichotomized from the general average. Biochemical assessments are specifically described below in graph form.    The results are expressed as the mean + SD. * Differences between ostomy patients versus health controls. p ≤ 0.05 were considered to be statistical significant from T test.

Figure 2.
Cortisol level in serum from ostomy patients compared to health controls. The results are expressed as the mean + SD. * Differences between ostomy patients versus health controls. p ≤ 0.05 were considered to be statistical significant from T test.

DISCUSSION
The investigation of depression, peripheral neurotrophic and cortisol parameters in ostomy patients is still poorly described. This is the first study to provide evidence of a significant BDNF decrease and NT-3 increase in the serum from ostomy patients, when compared to control group. These alterations were associated with psychological and social complications. In addition, the data showed an increase only in the HDRS, which is a scale for measuring the depressive symptoms levels, the SCID-I instrument is already used for diagnosing psychiatric disorders. Thus, ostomy patients did not have a MDD diagnosis, but presented a depressive symptoms improvement when compared to healthy controls 25 .
The main ostomy patient issues after discharge are: peristomal skin irritation, pouch leakage, odor, reduction of previously appreciated activities and depression or anxiety 5 . In clinical trials, it is possible to see that stoma use significantly reduces the QOL, and presenting depressive symptoms after the stoma surgery according to ostomy site 8 . In a study with 576 rectal cancer survivors, it was showed a frequent report of depression among rectal cancer survivors, especially in permanent ostomy patients, being associated with a financial expense increase. The overall prevalence of depression was 24% among rectal cancer survivors with the highest prevalence among those with a previous permanent ostomy (31%) 7 . The patient disease perception and coping influence the QOL and health due to self-efficacy, anxiety and depression 6 . Poor disease perception is related to increased anxiety, depression and reduced health -QOL, specifically by affecting sexuality and self-body image, social function and functional capacity, stomatal function and financial concerns 26 . A study with 500 inflammatory bowel disease patients (12.3% underwent ostomy) four factorial profiles were identified through exploratory factor analysis: somaticaffective, low self-esteem, suicidal and anhedonia. It was also identified that inflammatory markers and disease activity are significant predictors of somatic-affective symptoms and anhedonia 27 . In addition to this symptomatic profile categorization, it is possible to suggest that psychological distress can have a clinically relevant impact on wound healing, and depression is also associated with the healing impairment 28 . Therefore, we may suggest that this decrease in quality of life in ostomized patients may be a possible explanation for the depressive symptoms observed in the present study.
In the present study, we found no cortisol level alterations in ostomy patients when compared to health control group. Cortisol is secreted in response to stress and playing a key role by maintaining the body's blood pressure, immune function and anti-inflammatory processes 29 . The damage may occur when there is continuous or long-term stress exposure, resulting in continued cortisol production, leading to higher cortisol levels, which is a MDD risk 30 . Although, cortisol level alterations is not a diagnostic exam for MDD, but is a marker for hypothalamus-pituitary-adrenal (HPA) alteration 31 . Therefore, despite ostomy patients have depressive symptoms, seems that they not show HPA alteration.
The present study demonstrated an increase in depressive symptoms in ostomized patients when compared to healthy controls, accompanied by alterations in neurotrophins. Depression has been associated with disturbances in structural and synaptic plasticity 10 . Neurotrophic factors mediate several cellular functions through the receptor activation, including the gene expression that are integrally involved in the neuroplasticity regulation and cellular health. Most neurotrophin functions are mediating by the tropomyosin-related receptor tyrosine kinase (Trk) 21 . Studies have suggested that depressive mood results from the brain's inability to making appropriate adaptive responses to environmental stimuli as result of synaptic plasticity and impaired structural plasticity 32 .
The present study showed a BDNF decrease and NT-3 increase levels in serum from ostomy patients when compared to healthy controls. Several studies suggest that BDNF is involved in depression 33,34 . Previous study showed that depressed patients present decreased BDNF expression, and antidepressants up-regulate the BDNF expression 35 . It is well described that depressed patients have alterations in BDNF expression in blood cells [35][36][37] . Also, Ozan et al. 36 demonstrated that patients with MDD had a lower BDNF level in serum when compared to healthy control. In another study with 112 women post-treatment breast cancer survivors, the author evaluated depressive symptoms persist after the treatment completion and the Met allele of the BDNF Val66met polymorphism, suggesting these changes were activated via inflammatory processes arising from the treatment. Thus, BDNF Met allele could be a risk and vulnerability factor for inflammation-induced depression 38 . Suggesting an important role of inflammation in both depressive symptoms and changes in BDNF in this present study.
Hock et al. 39 used cerebrospinal fluid from elderly MDD patients, and compared to Alzheimer's disease ones, and mentally healthy control subjects. The results showed that NT-3 had a significantly increase in MDD patients than Alzheimer's disease patients and controls, suggesting that NT-3 increase may indicate a disturbance of the central noradrenergic system in these MDD patients. In another study, in drug-free subjects with bipolar disorder during manic and depressive episodes, the author showed increased in NT-3 40 . Further research is needed to better understanding this possible negative correlation between both neurotrophins.
In present study, the results did not demonstrate significant differences in NGF levels in serum from ostomy and control group. In a previous study, Kheirouri et al. 41 measure serum levels of BDNF and NGF in patients in first MDD episode compared to health controls. Their results showed BDNF decrease in depressive patients, and did not significant differences in NGF levels; NGF levels were not associated with stress, severity, or duration of the depressive episode.

CONCLUSION
In conclusion, the present study suggests ostomy triggers depressive symptoms and alterations in neurotrophins observed in blood collected from these patients.

INDIVIDUAL CONTRIBUTIONS
Valvassori, Schwalm, Ceretta and Jornada designed the study and wrote the protocol. Bavaresco, Rosa and Schwalm, managed the literature searches and analyses. Quadros, Zimmermann and Simon undertook the experimental procedures, biochemical and statistical analysis. Bavaresco and Quadros wrote the first draft of the manuscript. All authors contributed to and have approved the final manuscript.