Introduction

F1000Research

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F1000 Research Limited

London, UK

10.12688/f1000research.127917.1

Research Article

Articles

BDNF as a potential predictive biomarker for patients with pediatric cerebral palsy

[version 1; peer review: 1 approved with reservations]

Hanna

Hanan

Conceptualization Methodology Writing – Original Draft Preparation Writing – Review & Editing https://orcid.org/0000-0002-4463-3921 a 1 Youness

Eman R.

Conceptualization Writing – Original Draft Preparation Writing – Review & Editing https://orcid.org/0000-0002-6492-1680 2 Orban

Hisham A. Aziz

Conceptualization Data Curation Formal Analysis Writing – Review & Editing 2 El-Bassyouni

Hala T.

Conceptualization Formal Analysis Supervision Writing – Review & Editing 3 1Chemical Pathology, Faculty of Medicine, Delta University for science and Technology, Gamasa, 7731168, Egypt 2Medical Biochemistry Department, National Research Centre, Giza, 12622, Egypt 3Clinical Genetics Department, Human Genetics and Genome Research Institute, National Research Centre, Giza, 12622, Egypt

a hananagm@hotmail.com

No competing interests were disclosed.

18 11 2022

2022

1347

11 11 2022

2022

This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background: Cerebral palsy (CP) is the most common motor disability in children, which is instigated by damage to the developing brain that affects the ability to control the muscles. The main types of CP are spastic CP, dyskinesia CP and mixed CP. The aim of this work was to estimate the concentrations of complete blood count (CBC), erythrocytic sedimentation rate (ESR), C-reactive protein (CRP), brain-derived neurotrophic factor (BDNF), and tumor necrosis factor-α (TNF-α) in children with CP compared to the control group.

Methods: A total of 75 Egyptian children were enrolled in this study, 45 had CP and 30 were controls. CBC, ESR, CRP, BDNF, and TNF-α were assessed.

Results: The ESR, CRP and TNF-α levels showed statistically significant increases in cases compared with controls. While the neutrophil/lymphocyte ratio and the BDNF levels were significantly lower in CP compared with the controls. When comparing the different groups of CP with each other; there were no significant differences. Regarding the correlation of BDNF and different studied parameters, our study showed a positive correlation between BDNF and TNF levels only within the group with spastic CP.

Conclusions: BDNF may be considered as a biomarker or treatment target for CP to avoid further complications as still there is insufficient progress in the prediction, early diagnosis, treatment, and prevention of CP. Furthermore, searching for novel strategies to increase BDNF levels may open a new opportunity for the treatment of CP.

Cerebral palsy BDNF TNF-α ESR CRP

The author(s) declared that no grants were involved in supporting this work.

Introduction

Cerebral palsy (CP) is one of the most obvious forms of physical incapacity in childhood; with an estimated worldwide pervasiveness of around 1.5–3 per 1,000. ¹ CP is a wide umbrella term that covers lesions of the developing brain causing diminished movement and may affect perceptual and even cognitive problems. ² CP is usually confined to a group of disorders affecting posture and movement, causing limitation of activity and non-progressive turbulences, which usually happens during infant or fetal brain development. It was first termed ‘cerebro-spinal disorder’, and soon transitioned to the term cerebral palsy. ³ However, in spite of the change in the clinical diagnosis of CP, the definition continued to focus on the brain without stressing the developmental spinal cord dysfunction. ⁴

In regard to clinical overview, it begins in early childhood and persists throughout the individuals lifetime. ² Children with CP might develop a variety of secondary complications over time that will impact their functional capabilities showing huge variation from one another. ⁵ Complications may include feeding difficulties; pain; fractures; osteoporosis; communication difficulties; drooling; spasticity and contractures; osteopenia; and functional gastrointestinal abnormalities, vomiting, and constipation. ⁶ The associated intellectual impairment in children with CP needs more recognition, ⁷ as intellectual disability is an important and relatively common issue that influences daily activities, the burden of care, and of course quality of life. ⁸

The etiology of CP is complicated. For more than 100 years, it was speculated that the majority of CP cases were related to infant brain hypoxia in the perinatal period or during labor, which made CP incidence a matter of obstetric and neonatal care quality. CP is a result of heterogeneous risk factors complementary to specific etiology giving a wide variation in associated secondary conditions, functional limitations, severity, clinical features, and evolution of the condition over the lifetime of the person. ⁹ One of the most highlighted risk factors is premature birth, especially before 28 weeks of gestation, which has become one of the most associated risk factors for the development of CP. ¹⁰ It has long been thought that CP is caused by environmental triggers, recent findings identified rare mutations in single genes that may be the cause of this disorder. ¹¹ However, despite all the medical care done in these areas, CP incidence has remained unchanged. ¹²

Surveillance of Cerebral Palsy in Europe (SCPE), aiming to standardize CP classification has suggested a simple classification for patients, dividing them into three chief groups: ataxic, dyskinetic (choreoathetosis & dystonia), spastic (unilateral or bilateral spastic). ¹³ While mixed type is a combination of the aforementioned types. Another classification rendering to data from the Gross Motor Function Classification System (GMFCS): level 1, the patients are able to walk freely; level 2, patients walk with certain (slight) limitations on their own; level 3, patients walk by ancillary apparatus; level 4, patients could move on their own, but within specific limits, she/he could use an electrical wheelchair; level 5, patients are not capable to move on their own in a wheelchair. ¹⁴

As for the diagnosis of CP, it is fundamentally based on clinical findings. Early diagnosis is usually based on using standardized neuromotor assessment, findings on magnetic resonance imaging (MRI), and clinical history, moreover, in the greatest clinical settings, CP can only be diagnosed with confidence by the age of 2 years old. ¹⁵

Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophins (NTs), a family of proteins that support the function of the central nervous system (CNS), and it was recognized in 1982, as a small dimeric protein. ¹⁶ There are two main types of BDNF present in the human body, pro- and mature BDNF. ¹⁷ NTs are synthesized mainly in the CNS. ¹⁸ BDNF plays a crucial part in the development of the nervous system by acting on neuronal development, growth and survival, neurogenesis, cell differentiation, synaptic plasticity and synaptogenesis. ¹⁹ BDNF is expressed widely in the brain, comprising the hippocampus, cortex, and basal forebrain regions. It crosses the blood-brain barrier in a bi-directional mode and can therefore be detected in the blood. ²⁰ Pro-BDNF promotes neuronal cell death whereas mature BDNF stimulates neuronal cell survival and growth. ²¹ Differentiating between the BDNF forms has been well-known as one of the significant factors skewing reliable BDNF measurements, causing contradictory findings. ²² Along with the nervous system, BDNF is also found peripherally in the skeletal muscle, lungs smooth muscle cells and heart. The chief reservoir of this protein within the peripheral blood are platelets. ²³

Furthermore, the maximum concentrations of pro-BDNF are detected perinatally, following which it diminishes with age, while in adulthood it remains detectable at very low levels. These data justify partially that the brains of infants and newborns are more fragile to ischemia owing to lack of sufficient mature BDNF in the CNS and low-frequency neuronal activities. ²⁴

We aimed to assess the circulating concentrations of BDNF, TNF-α, and other blood parameters in children with CP compared with the control group. We then further subdivided CP groups according to clinical neurological affection into spastic, dyskinesia, and mixed groups, comparing the different biomarkers, including the blood level of BDNF, tumor necrosis factor-α (TNF-α), and other parameters with the control group.

Methods Ethics approval and consent

The goal of the study was clarified to the guardians of all participants. Written informed consent was obtained from all the parents of the children before they were enrolled in this study, which was permitted by the Ethical Committee, Faculty of Medicine, Delta University of Science and Technology, Egypt (FM2108007) in July 2021.

Participants

A total of 75 Egyptian children were enrolled from October 2021 to March 2022; CP was confirmed by clinical symptoms assessed by a neurological pediatrician. The CP children were enrolled as they were seeking physical therapy and follow up with pediatric clinicians. A total of 45 children with CP and 30 controls were enrolled; and they were matched by age, sex and socioeconomic status. The ages varied from 2 to 7 years old (mean age of controls: 3.9 ± 1.5, mean age of cases: 4.3 ± 1.5). As for control participants, they were selected by pediatric clinicians to be eligible for our study. They were enrolled following physical examination if they were determined to not show any symptoms or signs of active infection or chronic disease.

Participants were classified into two main groups. Group І comprised the controls, Group II included the participants with CP, which was further divided into three groups, spastic CP (26 patients), dyskinesia CP (12 patients) and mixed CP (seven patients) ( Physiopedia). ²⁵

Study design

Exclusion criteria of the CP patients and healthy volunteers included neurological, inflammatory, endocrine, cardiovascular, hepatic, renal or any clinically significant chronic disease and immunocompromised subjects. To all participants the following were done: 1.

Detailed history taking: detailed history was taken from all participants including starting of showing any delayed milestones.

Complete physical examination: complete physical examination was done for all groups.

Anthropometric measurements: namely body weight, height measurements.

Biochemical assessments

Assessment of liver function

Liver function enzyme alanine aminotransferase (ALT) was estimated using fully automated chemistry analyzer Cobas-C and Cobas. ²⁶

Assessment of C-reactive protein (CRP)

CRP was assessed using a fully automated Nephelometric quantitative technique, which is more specific than Latex. The analyzer includes a dilutor with temperature controlled (37°C) transfer arms; reagent, sample, standards rack stations (with barcode reading); buffer compartment; dilution racks; temperature controlled (37°C) cuvette rotor; cuvette washing device; bar code wand reader; and optical system. ²⁷

Assessment of erythrocytic sedimentation rate (ESR)

Using the Westergren method, which measures the distance (in millimeters) that red blood cells in anticoagulated whole blood fall to the bottom of a standardized, upright, elongated tube due to gravity over one hour, ERS was determined. ²⁸

Complete blood count (CBC)

CBC was analyzed using an automated blood cell counter (XE 2100). Haemoglobin; HBG, Neutrophil/lymphocyte ratio (NLR) was evaluated as the absolute neutrophil count divided by the absolute lymphocyte count, while the platelet lymphocyte ratio (PLR) was calculated by platelet count divided by absolute lymphocyte count. ²⁹

Quantification of TNF-α

TNF-α levels in serum was measured using a Human TNF alpha ELISA Kit (Abcam Cat# ab181421, RRID:AB_2924775).

Quantification of BDNF

BDNF levels in serum were measured using a Human BDNF ELISA Kit (Abcam Cat# ab212166, RRID:AB_2924770).

Both ELISA tests were done according to the manufacturer’s instructions. The full protocol can be found at protocols.io.

Sample number

Assuming that the confidence interval (CI) = 95% and the error = 5%, a cross-sectional study based on the previous situation in literature, was 8% among children (5–15 years old). Knowing that the average incidence of CP is estimated to range between 1.5 and 3.0 per 1,000 live births in Dakahalia, the estimated sample size to achieve 95% confidence and avoid α error, was calculated ( Calculator.net).

Statistical analysis of the data

Data were analyzed using IBM SPSS Statistics (RRID:SCR_016479) software package version 20.0 (Armonk, NY: IBM Corp). Checking for normality was performed by the Shapiro-Wilk test. Quantitative data were expressed as a range from minimum to maximum, mean, standard deviation, and median. For normally distributed quantitative variables, Student t-test was used to compare two groups, while one-way ANOVA test was used for comparing the four studied groups and followed by a post hoc test (Tukey) for pairwise comparison. The significance of the obtained results was judged at the 5% level. A Pearson correlation coefficient was performed between BDNF and TNF with different parameters.

Results

A total of 75 subjects were involved in our work ( Table 1). ⁴⁵ Their age ranged from 2 to 7 years old. A total of 35 patients were male (46.6%), and 40 were female (53.3%). No significant differences between the control group and patients regarding age, sex, weight, and ALT were found ( Table 1).

Table 1. Comparison between the two studied groups according to demographic data and laboratory investigation.

Parameter	Control (n = 30)	Cases (n = 45)	p-value
Age (years)
Mean ± SD.	3.9 ± 1.5	4.3 ± 1.5	0.280
Median (Min. – Max.)	3.5 (2 – 7)	4 (2 – 7)	0.280
Sex
Male	15 (50%)	20 (44.4%)	0.637
Female	15 (50%)	25 (55.6%)	0.637
Weight (kg)
Mean ± SD.	18.4 ± 2.3	18.5 ± 2.6	0.849
Median (Min. – Max.)	18.5 (14 – 23)	19 (13 – 24)	0.849
ALT (U/L)
Mean ± SD.	20.1 ± 4.8	19.6 ± 4.6	0.674
Median (Min. – Max.)	19 (12 – 29)	19 (13 – 29)	0.674
CRP (mg/l)
Mean ± SD.	1.7 ± 0.3	10.9 ± 2.6	<0.001 ^*
Median (Min. – Max.)	1.7 (1.2 – 2.2)	10 (8 – 19)	<0.001 ^*
TNF (pg/ml)
Mean ± SD.	16.4 ± 1.2	28 ± 3.6	<0.001 ^*
Median (Min. – Max.)	16 (15 – 18)	28 (14 – 34)	<0.001 ^*
ESR (mm/hr)
Mean ± SD.	7.9 ± 1.5	27.2 ± 3.9	<0.001 ^*
Median (Min. – Max.)	8 (5 – 10)	27 (20 – 35)	<0.001 ^*
HBG g/dl
Mean ± SD.	11.1 ± 0.6	12 ± 0.9	<0.001 ^*
Median (Min. – Max.)	11 (10.3 – 12.8)	11.9 (10.5 – 13.5)	<0.001 ^*
WBCs (10 ³ /ul)
Mean ± SD.	6865.3 ± 728.1	4981.1 ± 494.3	<0.001 ^*
Median (Min. – Max.)	6875 (4590 – 7900)	5100 (4150 – 5790)	<0.001 ^*
BDNF (pg/ml)
Mean ± SD.	30.7 ± 4.2	14.1 ± 2.5	<0.001 ^*
Median (Min. – Max.)	30 (23 – 38)	13 (10 – 18)	<0.001 ^*

SD: Standard deviation; t: Student t-test; χ ²: Chi square test; p: p value for comparing between the studied groups.

Statistically significant at p ≤ 0.05. ALT, alanine aminotransferase; CRP, C-reactive protein; TNF, tumor necrosis factor; ESR, erythrocytic sedimentation rate; HBG, hemoglobin; WBCs, white blood cells; BDNF, brain-derived neurotrophic factor.

The ESR, CBC, hemoglobin, lymphocyte, and NLR showed a statistically significant difference between the control group and cases ( Table 2).

Table 2. Comparison between the two studied groups according to CBC.

Parameter	Control (n = 30)	Cases (n = 45)	p-value
Neutrophils (10 ³ /ul)
Mean ± SD.	4,245.5 ± 434.8	2,654.8 ± 244.1	<0.001 ^*
Median (Min. – Max.)	4,180 (2,988 – 4,988)	2,670 (2,250 – 3,090)	<0.001 ^*
Neutrophils, %
Mean ± SD.	61.9 ± 2.1	53.4 ± 2	<0.001 ^*
Median (Min. – Max.)	62 (58 – 65)	53 (50 – 58)	<0.001 ^*
Lymphocytes (10 ³ /ul)
Mean ± SD.	2,619.8 ± 344.4	2,326.3 ± 282.3	<0.001 ^*
Median (Min. – Max.)	2,750 (1,602 – 3,150)	2,400 (1,790 – 2,900)	<0.001 ^*
Lymphocytes, %
Mean ± SD.	38.1 ± 2.1	46.6 ± 2	<0.001 ^*
Median (Min. – Max.)	38 (35 – 42)	47 (42 – 50)	<0.001 ^*
Neutrophils/Lymphocytes
Mean ± SD.	1.6 ± 0.2	1.1 ± 0.1	<0.001 ^*
Median (Min. – Max.)	1.6 (1.4 – 1.9)	1.1 (1 – 1.4)	<0.001 ^*

SD: Standard deviation; t: Student t-test; U: Mann Whitney test; p: p value for comparing between the studied groups.

Statistically significant at p ≤ 0.05. CBC, complete blood count.

BDNF levels in the CP group (14.1 ± 2.5 (pg/ml)) were significantly lower (P < 0.001) than those of controls (30.7 ± 4.2 (pg/ml)). When comparing the different groups of CP with each other; there were no significant differences. On the other hand, comparing each subtype of CP with the control group showed a significantly higher value for the control group (P < 0.001) ( Tables 3 and 4).

Table 3. Comparison between the four studied groups according to demographic data and laboratory investigation.

Parameter	Control (n = 30)	Cases (n = 45)			p-value
Parameter	Control (n = 30)	Spastic (n = 26)	Dyskinesia (n = 12)	Mixed (n = 7)	p-value
Age (years)
Mean ± SD.	3.9 ± 1.5	4.1 ± 1.6	4.8 ± 1.7	4.1 ± 1.1	0.449
Median (Min. – Max.)	3.5 (2 – 7)	4 (2 – 7)	5 (2 – 7)	4 (3 – 6)	0.449
Sex
Male	15 (50%)	13 (50%)	5 (41.7%)	2 (28.6%)	^MCp= 0.779
Female	15 (50%)	13 (50%)	7 (58.3%)	5 (71.4%)	^MCp= 0.779
Weight (kg)
Mean ± SD.	18.4 ± 2.3	17.7 ± 2.8	19.6 ± 1.9	19.6 ± 1.9	0.098
Median (Min. – Max.)	18.5 (14 – 23)	17.5 (13 – 24)	19.5 (17 – 22)	19 (17 – 22)	0.098
ALT (U/L)
Mean ± SD.	20.1 ± 4.8	20.8 ± 5.1	18.1 ± 2.3	17.9 ± 5	0.256
Median (Min. – Max.)	19 (12 – 29)	20 (13 – 29)	18.5 (14 – 22)	18 (13 – 25)	0.256
CRP (mg/l)
Mean ± SD.	1.7 ± 0.3	11 ± 3.2	10.7 ± 1.6	10.6 ± 1.7	<0.001 ^*
Median (Min. – Max.)	1.7 (1.2 – 2.2)	10 (8 – 19)	11 (8 – 14)	11 (8 – 13)	<0.001 ^*
p ₀		<0.001 ^*	<0.001 ^*	<0.001 ^*
Sig. bet. grps.		p ₁ = 0.953, p ₂ = 0.949, p ₃ = 1.000
TNF (pg/ml)
Mean ± SD.	16.4 ± 1.2	26 ± 2.8	32.5 ± 1	28 ± 0.8	<0.001 ^*
Median (Min. – Max.)	16 (15 – 18)	26.5 (14 – 29)	32.5 (31 – 34)	28 (27 – 29)	<0.001 ^*
p ₀		<0.001 ^*	<0.001 ^*	<0.001 ^*
Sig. bet. grps.		p ₁ < 0.001 ^* , p ₂ = 0.064, p ₃ < 0.001 ^*
ESR (mm/hr)
Mean ± SD.	7.9 ± 1.5	28.2 ± 3.9	25.3 ± 3.4	27 ± 3.7	<0.001 ^*
Median (Min. – Max.)	8 (5 – 10)	29 (20 – 35)	25.5 (20 – 30)	27 (22 – 33)	<0.001 ^*
p ₀		<0.001 ^*	<0.001 ^*	<0.001 ^*
Sig. bet. grps.		p ₁ = 0.039 ^* , p ₂ = 0.810, p ₃ = 0.623
HBG (g/dl)
Mean ± SD.	11.1 ± 0.6	12 ± 0.8	12 ± 1	11.7 ± 0.9	<0.001 ^*
Median (Min. – Max.)	11 (10.3 – 12.8)	12 (10.5 – 13.5)	11.9 (10.5 – 13.5)	11.8 (10.5 – 12.9)	<0.001 ^*
p ₀		<0.001 ^*	0.008 ^*	0.248
Sig. bet. grps.		p ₁ = 0.999, p ₂ = 0.792, p ₃ = 0.892
WBCs (10 ³ /ul)
Mean ± SD.	6,865.3 ± 728.1	5,047.3 ± 512.6	4,951.7 ± 455.9	4,785.7 ± 500.3	<0.001 ^*
Median (Min. – Max.)	6,875 (4,590 – 7,900)	5,100 (4,230 – 5,790)	5,160 (4,200 – 5,500)	4,700 (4,150 – 5,350)	<0.001 ^*
p ₀		<0.001 ^*	<0.001 ^*	<0.001 ^*
Sig. bet. grps.		p ₁ = 0.968, p ₂ = 0.738, p ₃ = 0.938
BDNF (pg/ml)
Mean ± SD.	30.7 ± 4.2	14.8 ± 2.5	13.6 ± 2.7	12.3 ± 0.8	<0.001 ^*
Median (Min. – Max.)	30 (23 – 38)	14 (10 – 18)	12.5 (11 – 18)	12 (11 – 13)	<0.001 ^*
p ₀		<0.001 ^*	<0.001 ^*	<0.001 ^*
Sig. bet. grps.		p ₁ = 0.703, p ₂ = 0.271, p ₃ = 0.835

SD: Standard deviation; χ ²: Chi square test; MC: Monte Carlo; F: F for One way ANOVA test, Pairwise comparison bet. each 2 groups was done using Post Hoc Test (Tukey); p: p value for comparing between the studied groups; p ₀: p value for comparing between Control and each other group; p ₁: p value for comparing between Spastic and Dyskinesia; p ₂: p value for comparing between Spastic and Mixed; p ₃: p value for comparing between Dyskinesia and Mixed.

Table 4. Comparison between the four studied groups according to CBC.

Parameter	Control (n = 30)	Cases (n = 45)
Parameter	Control (n = 30)	Spastic (n = 26)	Dyskinesia (n = 12)	Mixed (n = 7)	p-value
Neutrophils (10 ³ /ul)
Mean ± SD.	4,245.5 ± 434.8	2,674.2 ± 257.7	2,675 ± 199	2,548 ± 268.2	<0.001 ^*
Median (Min. – Max.)	4,180 (2,988 – 4,988)	2,655 (2,250 – 3,090)	2,760 (2,290 – 2,950)	2,450 (2,280 – 2,980)	<0.001 ^*
p ₀		<0.001 ^*	<0.001 ^*	<0.001 ^*
Sig. bet. grps.		p ₁ = 1.000, p ₂ = 0.814, p ₃ = 0.856
Neutrophils, %
Mean ± SD.	61.9 ± 2.1	53.1 ± 2	54.1 ± 2	53.1 ± 2	<0.001 ^*
Median (Min. – Max.)	62 (58 – 65)	53 (50 – 58)	54 (51 – 58)	52 (51 – 56)	<0.001 ^*
p ₀		<0.001 ^*	<0.001 ^*	<0.001 ^*
Sig. bet. grps.		p ₁ = 0.495, p ₂ = 1.000, p ₃ = 0.767
Lymphocytes (10 ³ /ul)
Mean ± SD.	2,619.8 ± 344.4	2,373.1 ± 288.4	2,276.7 ± 279.6	2,237.7 ± 265.9	0.001 ^*
Median (Min. – Max.)	2,750 (1,602 – 3,150)	2,435 (1,790 – 2,900)	2,390 (1,900 – 2,700)	2,250 (1,840 – 2,564)	0.001 ^*
p ₀		0.020 ^*	0.009 ^*	0.022 ^*
Sig. bet. grps.		p ₁ = 0.809, p ₂ = 0.734, p ₃ = 0.993
Lymphocytes, %
Mean ± SD.	38.1 ± 2.1	46.9 ± 2	45.9 ± 2	46.9 ± 2	<0.001 ^*
Median (Min. – Max.)	38 (35 – 42)	47 (42 – 50)	46 (42 – 49)	48 (44 – 49)	<0.001 ^*
p ₀		<0.001 ^*	<0.001 ^*	<0.001 ^*
Sig. bet. grps.		p ₁ = 0.495, p ₂ = 1.000, p ₃ = 0.767
Neutrophils/Lymphocytes
Mean ± SD.	1.6 ± 0.2	1.1 ± 0.1	1.2 ± 0.1	1.2 ± 0.1	<0.001 ^*
Median (Min. – Max.)	1.6 (1.4 – 1.9)	1.1 (1 – 1.4)	1.2 (1 – 1.4)	1.1 (1.1 – 1.3)	<0.001 ^*
p ₀		<0.001 ^*	<0.001 ^*	<0.001 ^*
Sig. bet. grps.		p ₁ = 0.390, p ₂ = 0.697, p ₃ = 0.778

SD: Standard deviation; F: F for One way ANOVA test, Pairwise comparison bet. each 2 groups was done using Post Hoc Test (Tukey); H: H for Kruskal Wallis test, Pairwise comparison bet. each 2 groups was done using Post Hoc Test (Dunn's for multiple comparisons test); p: p value for comparing between the studied groups; p ₀: p value for comparing between Control and each other group; p ₁: p value for comparing between Spastic and Dyskinesia; p ₂: p value for comparing between Spastic and Mixed; p ₃: p value for comparing between Dyskinesia and Mixed.

Statistically significant at p ≤ 0.05. CBC, complete blood count.

The TNF level in the CP group (28 ± 3 (pg/ml)) was significantly higher (P < 0.001), than those of the control group (16.4 ± 1.2 (pg/ml)). When comparing the different groups of CP with each other; there was a significant difference between the group with Spastic CP (26 ± 2.8 (pg/ml)) and dyskinesia (32.5 ± 1 (pg/ml)) (P < 0.001). Moreover, when comparing Dyskinesia CP (32.5 ± 1 (pg/ml)) and mixed CP (28 ± 0.8 (pg/ml)) there was a significant difference (P < 0.001), with higher levels found in the Dyskinesia group. On the other hand, comparing spastic CP and dyskinesia CP showed no significant difference (p ₂ = 0.064) ( Tables 3 and 4).

Furthermore, the CRP level was significantly higher in the CP group (10.9 ± 2.60 (mg/l)), than those of the control group (1.7 ± 0.3 (mg/l)) (P < 0.001). While comparing the different groups of CP with each other; there was no significant difference. On the other hand, comparing each subtype of CP with the control; it showed a significantly higher value for the control group (P < 0.001) ( Tables 3 and 4).

Also, the NLR was significantly lower in the CP group (1.1 ± 0.1), than those of the control group (1.6 ± 0.2) (P < 0.001). When comparing the different groups of CP with each other; there was no significant difference. On the other hand, comparing each subtype of CP with the control; it showed a significantly higher value for the control group (P < 0.001) ( Tables 2 and 4).

With regard to the correlation of BDNF with the various studied parameters, our study only showed a positive correlation between BDNF and TNF levels with spastic CP ( Table 5), otherwise there were no significant correlations with other parameters ( Table 6).

Table 5. Correlation between BDNF with different parameters.

BDNF (pg/ml) vs.	Control (n = 30)		Cases (n = 45)		Spastic (n = 26)		Dyskinesia (n = 12)		Mixed (n = 7)
BDNF (pg/ml) vs.	r	p	r	p	r	p	r	p	r	p
CRP (mg/l)	0.210	0.265	-0.170	0.263	-0.241	0.236	-0.242	0.448	0.238	0.607
Neutrophils	0.165	0.383	-0.106	0.487	-0.202	0.323	-0.072	0.825	-0.651	0.113
Lymphocytes	0.007	0.972	-0.122	0.426	-0.308	0.126	0.010	0.975	-0.530	0.221
N/L ratio	0.208	0.271	-0.015	0.923	0.189	0.356	-0.167	0.604	-0.258	0.576
TNF (pg/ml)	0.002	0.992	0.015	0.923	0.398	0.044 ^*	0.050	0.877	0.000	1.000

r: Pearson coefficient; N/L ratio: Neutrophils/Lymphocytes.

Statistically significant at p ≤ 0.05. BDNF, brain-derived neurotrophic factor; CRP, C-reactive protein; TNF, tumor necrosis factor.

Table 6. Correlation between TNF with different parameters.

TNF vs.	Control (n = 30)		Cases (n = 45)		Spastic (n = 26)		Dyskinesia (n = 12)		Mixed (n = 7)
TNF vs.	r	p	r	p	r	p	r	p	r	p
CRP (mg/l)	0.038	0.840	-0.194	0.201	-0.322	0.109	0.394	0.205	0.594	0.160
N/L ratio	0.093	0.627	0.163	0.283	-0.080	0.699	0.560	0.058	-0.418	0.350

r: Pearson coefficient; N/L ratio: Neutrophils/Lymphocytes; TNF, tumor necrosis factor.

Discussion

CP is mainly a neuromotor disorder that affects the muscle tone, posture and development of movement. ³⁰ Precise CP diagnosis needs either an abnormality on an MRI scan and/or clinical history and motor dysfunction to ensure long-term functional outcomes based on positively modulating neuroplasticity. ³¹

NTs are a family of proteins, which include BDNF, that are actively produced throughout the brain and are implicated in neuronal activity regulation; they also help and stimulate neurogenesis, which is why they are known to play an important role in both the recovery and protective function following neurodegenerative diseases. ³²

Many researchers have been dedicated to elucidating its role in pathological and physiological conditions. Multiple factors give complexity to the BDNF system, i.e., different receptors with their multiple signaling pathways, the functional isoforms and heterogeneous population of mRNAs, demanding more accurate procedures to make field improvements. ³³

In our study, the level of BDNF was significantly lower in the CP group compared with the control group. Hansen et al., stated that the reduced level of BDNF in the CP group was observed in children with severe CP, specifically low intake of n-3 fatty acids, energy, and dietary fibers and low physical activity levels. ³⁴ BDNF plays a great role in the development of the nervous system by affecting synaptogenesis, neurogenesis, neuronal development, growth and survival, synaptic plasticity and cell differentiation. ¹⁹

AL-Ayadhi, suggested in her study a highly possible pathophysiological role played by BDNF in autism, as they also found significantly lower BDNF levels in participants with autism but it was not related to the severity level, this correlation may interpret the pathogenesis of autism and other possible neurological dysfunctions. ³⁵

Forsgren et al., ³⁶ found a correlation between BDNF and TNF levels in patients with rheumatoid arthritis (RA), they stated that the level of BDNF was lowered significantly with anti-TNF treatment, which was associated with pain relief. Likewise, we found only a positive correlation between BDNF and TNF levels with spastic CP.

On the other hand, Siang Ng et al., suggested in their study that BDNF is increased in mild cognitive impairment (MCI) cases as a compensatory mechanism in preclinical dementia, supporting the partially inflammatory and neurotrophic hypotheses of cognitive impairment. ²² Plasma BDNF appears to be a useful biomarker to differentiate MCI from patients with no cognitive impairment. This supports the suggestion that augmented peripheral BDNF acts as a compensatory mechanism in the preclinical stage of dementia. ³⁷

Palasz et al., suggested that NTs has been demonstrated that synaptic plasticity defects associated with insufficient neuronal supply of BDNF and other neurotrophic factors may contribute to neurodegenerative and neuropsychiatric diseases as in Parkinson’s and Alzheimer’s disease. ³⁸ Nevertheless, neither efforts to enhance BDNF expression with gene therapy nor direct delivery of exogenous BDNF into the patient’s brain led to any improvements.

Yeom et al., stated that a high peripheral BDNF concentration was found in preschool aged children with cognitive and attention intellectual disabilities, suggesting that it may help as a biomarker for any intellectual problems in early life. ³⁹

Weinstein et al., found that older adults with higher peripheral BDNF levels had lower chance of developing Alzheimer’s disease. ²¹ Decreased BDNF levels may explain the lack of trophic support, contributing to neuronal degeneration. ⁴⁰

Furthermore, it has been shown that neurodegeneration and psychiatric diseases may be partially affected by low levels of BDNF and other members of the NT family. ⁴¹ Therefore, a search is needed for new strategies to increase the levels of BDNF as a tool in the treatment and prevention of neurological diseases. ³⁸

Wu J et al., revealed that children with cerebral palsy have greater levels of TNF-α than controls ⁴²; this corresponded to our findings, in addition, they suggested that pharmaceutical therapies to reduce inflammation in infants with cerebral palsy may alleviate symptoms and warrant further investigation. Increased baseline TNF-α levels might be an indicator of more extensive brain damage when correlated with severity.

Also, it was suggested that the inflammatory response might diminish with age, whereas the cerebral palsy symptoms are not necessarily improved. ⁴³ Similarly, Soliman S et al., concluded that TNF-α levels in children with spastic CP are positively correlated with disease severity, showing a significant difference between pretreatment levels of TNF-α, which improved after rehabilitation and correlated with improvement in all outcome measures. ⁴⁴

Conclusions

BDNF has been shown to have a significant relationship with brain development early in life. However, further studies are needed to assess its value as a biomarker and/or as a treatment to enhance or avoid further complications of CP as still there is insufficient progress in prediction, early diagnosis, treatment, and prevention. Searching for novel strategies to increase BDNF levels may open a new opportunity for the treatment of neurological diseases.

Data availability Underlying data

Open Science Framework: BDNF as Predictor biomarker for Pediatric Cerebral Palsy patients. https://doi.org/10.17605/OSF.IO/5BKAE. ⁴⁵

Data are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).

Acknowledgments

We appreciate all patients who participated and contributed samples to the study.

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Reviewer response for version 1

Gouveia

Henrique

1 Referee https://orcid.org/0000-0002-8784-3881 1Federal University of Pernambuco, Recife, Brazil

Competing interests: No competing interests were disclosed.

10 5 2024

2024

This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

recommendation

approve-with-reservations

In general, I suggest having the text proofread by a native speaker, as there are some grammatical errors.

Abstract

- Given that the abstract needs to present the work in a complete way, I suggest adding some information. In the background, a sentence before the objectives presenting what has been found in the literature about the parameters in the context of CP that were evaluated could better justify the selection of these parameters. In the methods, it would be interesting to add the number of each type of CP among the 45 individuals.

- I suggest writing the keywords in full and presenting the acronyms in the abstract, as presented.

Introduction

- Please check the study by McIntyre et al. (2022) [Ref 1]for more recent data on the prevalence of CP.

- The authors presented information on BDNF, but not on the other parameters. It is important to present something about the parameters that were evaluated to justify the evaluation, including data from conditions similar to CP that observed changes in these parameters. Otherwise, the selection of parameters seems to have been random.

Methods

- The enzyme alanine aminotransferase (ALT) had not been mentioned elsewhere before appearing in the methods, so it would be interesting to standardize the objectives in the methods and introduction.

Results

- In the description of the results in Table 1, as well as describing in the text the parameters that were not different, I suggest adding the differences found between the groups.

- General comment on the table legends: In the legend for table 1, the authors have indicated that “t: Student t-test; χ2: Chi square test”, should they be somewhere in the table? If they don't, just say that the statistical test was used without stating what “t and χ2” mean, as they won't be present in the table. This also applies to “p: p value”, since the table says “p-value”. In other tables, such as 3 and 4, the authors presented “F: F for One way ANOVA test” and “H: H for Kruskal Wallis test”, but there is no F and/or H in the table. In general, I suggest that the authors revise the table legends and leave only the acronyms that were presented in the tables that need to be explained, otherwise just indicate the statistical test performed.

- In the description of the results in table 2, the authors reported differences in ESR and hemoglobin, but they were presented in table 1.

- In the description of the results in tables 3 and 4, the authors need to review the means and variances used to describe significant differences between the CP and Control groups, since these results were cited in table 1, and not in tables 3 and 4, which in turn present the specific results for each type of CP.

- The authors reported in the text that “comparing spastic CP and dyskinesia CP showed no significant difference” with regard to TNF. I believe the authors meant to refer to the comparison between spastic CP and mixed CP. The authors also cited table 4 in this and the previous paragraph (BNDF), but it does not show BDNF or TNF data, but CBC data.

- Table 3 also shows other differences that were not mentioned in the text, such as the difference in ESR at p1, and differences in ALT, HBG, and WBCs at p0. This table is different from table 1, which presents the differences only between CP and Control, so the differences need to be presented in the text.

- Table 4 was also cited incorrectly when describing the CRP results.

- The description of the results in table 4 needs to be more complete and not just for the NLR, as indicated in table 3.

- I suggest that the authors separate the description of tables 5 and 6 to make it clear that 5 is about BDNF and 6 about TNF. Also, why did the authors decide to correlate TNF only with CRP and N/L ratio and not make the same comparisons made with BDNF in table 5?

Discussion

- I suggest that the authors start the discussion by presenting a summary of the results they found. In addition, the authors focused the discussion only on BDNF and ignored all the other results. Thus, a more complete discussion needs to be done.

Is the work clearly and accurately presented and does it cite the current literature?

Partly

If applicable, is the statistical analysis and its interpretation appropriate?

I cannot comment. A qualified statistician is required.

Are all the source data underlying the results available to ensure full reproducibility?

Yes

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

Partly

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

Reviewer Expertise:

Nutrition and Neurosciences, with emphasis on the alterations caused by experimental Cerebral Palsy and possible nutritional and pharmacological interventions.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

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