Aedes genus. Zika virus (ZIKV) is part of this genus. It was initially reported in Brazil in December 2014 as an unknown acute generalized exanthematous disease and was subsequently identified as ZIKV infection. ZIKV became widespread all over Brazil and was linked with potential cases of microcephaly.Flaviviridae family belongs to the Spondweni serocomplex, which is mainly transmitted by vectors from the via ultrasonography, which was performed at 29.5 weeks of gestation. The patient presented with multiple abnormalities, which range from a suggested Arnold–Chiari malformation, compromising the lateral and third ventricles, liver calcifications, bilateral pyelocalic dilatations, other brain anomalies, and microcephaly. At 12 weeks of gestation, the vertical transmission of ZIKV was suspected. At 38.6 weeks of gestation, the newborn was delivered, with the weight in the 10th percentile , height in the 10th percentile (48 cm), and cephalic circumference under the 2nd percentile (31 cm). Due to the physical findings, brain magnetic resonance imaging (MRI) was performed, revealing a small and deviated brain stem, narrowing of the posterior fossa, a giant posterior fossa cyst with ventricular dilatation, a severe cortical and white matter thinning, cerebellar vermis with hypoplasia, and superior and lateral displacement of the cerebellum. In addition, hydrocephalus was displayed by the axial sequence, and the cerebral cortex was also compromised with lissencephaly. Schizencephaly was found with left frontal open-lip, and no intracranial calcifications were found. Two novel heterozygous nonsense mutations were identified using whole-exome sequencing, and both are located in exon 8 under the affection of ZIKV congenital syndrome (CZS) that produced a premature stop codon resulting in the truncation of the cyclin-dependent kinase 5 regulatory subunit-associated protein 2 (CDK5RAP2) protein.We report a case of a 28-year-old Colombian woman, who came to the Obstetric Department with an assumed conglomerate of fetal abnormalities detected CDK5RAP2 gene was also identified. The CDK5RAP2 gene encodes a pericentriolar protein that intervenes in microtubule nucleation and centriole attachment. Diallelic mutation has previously been associated with primary microcephaly.We used molecular and microbiological assessments to report the initial case of vertically transmitted ZIKV infection with congenital syndrome associated with a neurological syndrome, where a mutation in the  Aedes genus  were confirmed by reverse-transcription (RT)-polymerase chain reaction (PCR) assay. Furthermore, 11,944 pregnant women were reported as potential cases of ZIKV infection, but only 1,484 (12%) of these were confirmed. From this population, 50 newborns were reported as possible ZIKV congenital syndrome (CZS), but ZIKV infection was confirmed in only 4 (8%) of them .CDK5RAP2 gene encodes a pericentriolar protein that mediates microtubule nucleation and centriole attachment  was identified. The tachment , and diaIn mid-2016, a 28-year-old previously healthy Colombian woman with later prenatal care attention came to the Perinatology and Obstetric Department at Fundación Valle del Lili-University Hospital due to numerous fetal abnormalities observed in ultrasonography done at 29.5 weeks of gestation, which demonstrated brain anomalies with a possible Arnold–Chiari malformation, compromised lateral and third ventricles, liver calcifications, bilateral pyelocaliceal dilatations, microcephaly, and an apparent clubfoot. All relevant antecedents during pregnancy were considered . The patient had experienced an episode of illness for 2–7 days that was characterized by fever, myalgias, and vomiting. She went to the hospital, where ZIKV was presumed, and RT-PCR of the amniotic fluid was done, with a positive result at 12 weeks of gestation. The findings confirmed ZIKV vertical transmission and congenital infection. Inadequate prenatal follow-ups were performed until the fetal examination at 29.5 weeks, which showed multiple malformations.At the time of delivery, the mother was traveling from a remote area of the country. She went into labor on the way to the hospital and gave birth in the ambulance. According to the medical records and first ultrasonography date, delivery was at 38.6 weeks of gestation. The male newborn weighed 3,180 g (10th percentile), with a height of 48 cm (10th percentile) and a cephalic circumference of 31 cm (under the 2nd percentile) according to the WHO Growth Charts from the Centers for Disease Control and Prevention (CDC) . TransfoThe mother reported that the newborn was irritable, with an abnormal pattern of crying and hyperexcitability. The physical examination showed distal tremors, hypertonia, trunk hyperextension, spasticity, increased deep tendon reflexes, persistent primitive responses, clenched fists, strabismus, and nystagmus. Microcephaly was present, which was characterized by craniofacial disproportion, decreased vertical size of the skull, a sloping forehead, and pronounced supraorbital ridges, giving the appearance of oversized facial features and proptosis. Epicanthal folds, retrognathia, excess skin over the entire scalp and forehead, and occipital and nuchal skin folds were also found, giving the appearance of a short neck. The forehead presented with bilateral depressions and prominent overlapping metopic and sagittal sutures with supratemporal depressions and a large visible occipital prominence. An examination of the extremities revealed hand contractures, camptodactyly, feet contractures, prominent calcanei, multiple dimples, and arthrogryposis . BecauseAfter 1 year of follow-up, a complete checkup was performed. The findings showed irritability, an abnormal pattern of crying, hyperexcitability, hypertonia, spasticity, clenched fists, strabismus, clinodactyly of the fifth finger, and an ogival palate. Microcephaly was still present, with a few of the previously observed characteristics, such as a small skull, retrognathia, and a prominent metopic suture. A physical examination of the extremities found hand contracture and camptodactyly, feet contracture, prominent calcanei, multiple dimples, arthrogryposis, and a severe delay in the developmental milestones. A brain magnetic resonance imaging (MRI) showed microcephaly, with a giant posterior fossa cyst, ventricular dilatation, hypoplasia of the cerebral vermis, and an anomalous cerebral cortex with lissencephaly .Following the National Institutes of Health recommendations, amniocentesis was performed at 12 weeks of gestation for ZIKV infection case suspicions. Ultrasound-guided transabdominal amniocentesis was done, and around 5 ml of amniotic liquid was aspirated and instantly stored at −80°C for testing. Further samples were taken from the cerebrospinal fluid when the infant was born, and the blood, amniotic fluid, and placenta were also taken, as is previously described in The molecular identification of ZIKV in the amniotic fluid and cerebrospinal fluid was done by using a SuperScript III Platinum One-Step RT-PCR system  run by the Colombian Government National Institutes of Health using the primers ZIKV 1087, ZIKV 1163, and ZIKV 1108-FAM, followed by the Lanciotti protocol . For hybSamples were obtained from the mother and the newborn and those from viral isolation trials for ZIKV RNA, using a TaqMan RT-PCR assay and quantitative RT-PCR for ZIKV following the CDC protocol . StandarThe classical cytogenetics G-banding technique was performed after the patient had given birth to do a blood karyotype analysis. The karyotype analysis was performed on cells in metaphase using a microscope and the CytoVision (Leica) karyotype software system. After extracting DNA from the patient’s peripheral blood cells, 1 μg was used for array comparative genomic hybridization (a-CGH) utilizing KaryoArray 3.0  and was marked by fluorescence to compare the patient’s DNA with the control sample. DNA hybridization was done with a human genomic microarray of 860 K oligonucleotides using commercially available Agilent-based arrays, which were analyzed using an Agilent scanner with Feature Extraction 9.1 software. The aberration detection method 2 algorithm was used to determine any statistically significant aberrations. This was defined as the minimum number of oligonucleotides to consider an alteration. Subsequently, the medium resolution of the array was one oligonucleotide per 9 Kb in the regions of maximum interest, such as microdeletions and microduplications, centromeres, and telomeres. The algorithm in CGH Analytics version 3.5 software (Agilent Technologies) was used , as is dThe whole-exome sequencing (WES) was captured by using a SureSelect Human All Exon capture kit (Agilent) of 51 Mb, and sequencing was done by using an Illumina Hiseq 2000 sequencing system . During the sequencing, paired readings of 101 nucleotides in length were acquired. The different variants were analyzed by focusing on genes related to microcephaly. Only variants in the coding region and flanking intronic regions with minor allele frequency <1% were evaluated and compared using datasets acquired from the 1000 Genomes Project Consortium, dbSNP, Exome Variant Server, and Exome Aggregation Consortium databases. Variants in around 20 bp of flanking intron regions were examined and sequenced by Sanger sequencing . ParentaTwo heterozygous nonsense mutations in CDK5RAP2 were analyzed with variant functional prediction software tools  in order to predict the pathogenicity of the mutations. In addition, these variants were searched in Human Gene Mutation Database (HGMD), Leiden Open Variation Database (LOVD), and ClinVar database, as is described previously in the literature .® ND-1000 Spectrophotometer . RNA reverse transcription was performed by a High-Capacity cDNA Reverse Transcription Kit  using 100 ng of total RNA according to the manufacturer’s instructions. For quantitative PCR (qPCR), cDNA was amplified using CDK5RAP2 primers 5′-GTTGGGGAAATGGTCTGCTCC-3′ and 3′-TATGTTCAGTGGGGCCATGA-5′. PCR amplification was performed in a 20-μl volume that contained: 10 μl of the EXPRESS SYBR® GreenERTM qPCR SuperMixes (0.25×) kit , 0.4 μl of each of the primers (1×), 0.04 μl ROX (50 nM), 1 μl of cDNA, and 8.16 μl of water. Real-time PCR was performed in a 7500-Fast real time PCR instrument, and reaction was carried out with the following conditions: 95°C for 20 s, followed by 40 cycles of: 95°C for 1 s and 60°C for 20 s and for the melt curve: 95°C for 15 s, 60°C for 60 s, 95°C for 15 s, and 60°C for 15 s. GAPDH mRNA was used as an internal control (house-keeping gene), and the relative expression of each transcript was calculated using the 2–ΔΔCt method. RT-PCR reactions were performed for each sample in triplicate. For the set of reference samples, we used mRNA extracted from the patient, parents, and two additional healthy individuals . RNA integrity and concentrations were both analyzed using a 1% agarose gel and Nanodropividuals . Data arividuals .Written informed consent was obtained from the patient’s parents for the publication of the case details and accompanying images. All research was conducted according to the Declaration of Helsinki, and the research protocol was registered with the following number #253. This study received approval from the Ethics Committee of Fundación Valle del Lili (Act 11/2016).Serum and urine samples were tested for chikungunya virus, dengue virus, and ZIKV. The RT-quantitative PCR assays for dengue and chikungunya viruses were negative for whole samples and positive for ZIKV in the amniotic liquid of the patient but negative in the urine and serum samples from the fetus and mother. Serology tests of the serum and urine using anti-dengue virus IgM, anti-dengue-virus IgG, anti-chikungunya-virus IgM and IgG, and anti-ZIKV displayed negative results in the enzyme-linked immunosorbent assays (ELISAs). The samples also tested negative for TORCH , as is described previously in the literature .Fetal karyotyping displayed regular 46, XY  profiles. No losses and/or gains of genomic material were detected in the fetal DNA sample, as is described previously in the literature .CDK5RAP2 gene at positions p.Thr274LysfsTer27 and p.Glu275ArgfsTer16, respectively, when compared with the human reference template (GRCh38) or mutation databases (LOVD and HGMD). These variants result in a stop codon causing the early termination of the protein. Bioinformatic analysis was performed using the University of California Santa Cruz Genome Browser, which showed that the region was highly conserved between species and was located near to a regulatory element, such as H3K27Ac, and between a promoter and enhancer region. According to the AmiGO gene ontology browser and Kyoto Encyclopedia of Genes and Genomes, this gene has multiple functions, such as G2/M transition of the mitotic cell cycle, establishment of mitotic spindle orientation, microtubule cytoskeleton organization, maintaining centrosome function, spindle pole assembly and orientation, microtubule bundle formation, and protein binding . At the moment, they are classified as “probably pathogenic” according to the American College of Medical Genetics and Genomics guidelines see .Quantitative gene expression analysis using real-time PCR showed that mRNA levels of CDK5RAP2 were downregulated in the patient mouth swab sample. Our results suggest an approximately 0.6-fold decrease of CDK5RAP2 expression in the patient mouth swab sample compared with the control mouth swab sample .The term CZS was created to characterize the range of congenital abnormalities associated with ZIKV infection, including microcephaly to ocular injury and hearing loss . At the Recent studies have suggested that placental injury and infarctions might affect fetal oxygenation . FurtherASPM, CASC5, CENPF, MCPH1, RBBP8, STIL, and TBR2). These genes were shown to share putative homologs that were co-expressed with CDK5RAP2, and molecular interaction occurred between ASPM and CDK5RAP2  codes a protein that is located in the microtubules, especially at the growing ends, in interphase cells. The CDK5RAP2–MAPRE1 complex induces microtubule bundling and assembling, suggesting that this complex regulates microtubule dynamics. The mutations p.T274KTer27 and p.E275RTer16 produce a truncated protein with loss of the protein–protein interactions in the CDK5RAP2–MAPRE1 complex. This loss affects the self-assembling dynamics of the microtubules, resulting in the dysregulation of neuron production and brain size  found in this case, which is also related and required for the maintenance of the germ cell pool during embryonic development and is linked with molecular evolutionary pathways for regulating the brain size (Patients with autosomal-recessive primary microcephaly are widely known as having an isolated brain disorder without other system involvement. The present piece of evidence demonstrates the overlapping of the two syndromes on clinical manifestations ,2. The pain size . This inain size . In addiain size . FurtherCDK5RAP2 gene encodes a pericentriolar protein that mediates microtubule nucleation and centriole attachment. A diallelic mutation on this gene has been previously associated with primary microcephaly.In summary, our study describes a case of vertical ZIKV transmission associated with a congenital ZIKV syndrome and linked with a neurological syndrome, with microbiological, molecular, and genetic assessments, where two cis-novel variants in CDK5RAP2 were found. The The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.This study was approved by the Ethics Committee of Fundación Valle del Lili.All authors made a substantial contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas. They took part in drafting, revising, or critically reviewing the article and gave final approval of the version to be published. They have agreed on the journal to which the article has been submitted and agreed to be accountable for all aspects of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This study provides further evidence of CNS in some NMDs and reinforces the need to include cognitive assessment in protocols of care of selected pediatric NMDs.Central nervous system (CNS) involvement has been variously studied in pediatric neuromuscular disorders (NMDs). The primary goal of this study was to assess cognitive functioning in NMDs, and secondary aims were to investigate possible associations of cognitive impairment with motor impairment, neurodevelopmental delay, and genotype. This was a cross-sectional study of 43 pediatric patients, affected by six NMDs. Myotonic dystrophy type 1 (DM1) and glycogen storage disease type 2 (GSD2) patients had a delay on the Bayley-III scales. On Wechsler scales, DMD and DM1 patients showed lower FSIQ scores, with an intellectual disability (ID) in 27% and 50%, respectively. FSIQ was normal in Becker muscular dystrophy (BMD), GSD2, and hereditary motor sensory neuropathy (HMSN) patients, while higher individual scores were found in the spinal muscular atrophy (SMA) group. In the DM1 cohort, lower FSIQ correlated with worse motor performance (ρ = 0.84,  The last 20 years have witnessed a major expansion in the research and clinical management of neuromuscular disorders (NMDs), with an exponential increase in the knowledge of the basic biological-molecular mechanisms of illness and the introduction of innovative therapeutic applications. Considerable progress has been made in the development of causal, mechanism-based, and disease-modifying therapies, especially with the innovative application of genetic therapy technologies. For Duchenne muscular dystrophy (DMD) patients, steroids are now part of the recommended standard of care , and genThe new disease-modifying therapies, associated to appropriate standard of care, are improving the prognosis of patients and leading to the emergence of new clinical phenotypes , with moCognitive impairment, apparently independent from motor impairment , is recoLittle is known about the CNS involvement in BMD, and the type and prevalence of cognitive impairment has not been determined, even though these patients appear to have average cognitive functioning, with few reports of a slight reduction in verbal skills , learninCNS involvement is a core manifestation of DM1 ,20, but Patients with infantile-onset Pompe disease (IOPD) are now living longer since the advent of intravenous (ERT) . Brain aConcerning patients affected by SMA, with the increasing number of long-term survivors worldwide, new phenotypes are arising, suggesting that some patients may show cognitive impairment, in particular those with SMA type 1. Clinical practice shows that most of these untreated children never achieve functional verbal skills ,33. PoorRegarding HMSN, since it is a peripheral neuropathy, there is almost no literature concerning CNS involvement. A recent study published in 2017 has investigated a possible role of PMP22 protein (lacking in CMT1A) in CNS functioning: results of this study suggest that an altered expression of PMP22 determines a mild, but significant, ID and a reduction in the volume of white matter on MRI, compared to healthy subjects, but further studies in pediatric age are needed .The introduction of new treatments and the consequent improved survival and improvement in motor functions make the assessment of cognitive functioning especially timely and clinically relevant to the management of subjects with NMDs. This pilot study aimed to test the feasibility of evaluating CNS involvement through the assessment of neurocognitive functioning in children affected by genetic NMDs of different etiology and degrees of motor impairment. Possible associations between motor deficit and intellectual functioning, developmental delay and cognitive impairment, and genotype–phenotype correlations were also investigated. The study was intended as an initial step towards ultimately characterizing cognitive phenotypes and improving clinical management.This was a single-center, observational, cross-sectional study conducted at the child neuropsychiatry services of the University of Turin Pediatric Hospital “Regina Margherita”, Turin, Italy, over a 6-month period. Cognitive functioning was assessed in children and adolescents affected by 6 of the main NMDs followed at our center: DMD, BMD, DM1, GSD2, SMA type 2 and 3, and HMSN type 1 and 2. The study was approved by the institution’s research ethics committee and written informed consent to study participation was provided by parents or legal guardians.Patients aged below 17 years, with a diagnosis of NMDs made within the first 12 years of life, were enrolled. NMDs were diagnosed according to the current international standard criteria, based on clinical and laboratory characteristics with genetic-molecular confirmation.Patients were excluded if unable to adequately perform the whole cognitive assessment because of too severe disabling motor impairment or if unable to understand instructions during tests for linguistic reasons .Cognitive and motor function assessment scales, appropriate for age and pathology, were administered. Patients who had not undergone a cognitive assessment during their clinical history were recalled and assessed during the study period, while for those previously assessed, the results were retrieved retrospectively. Patients’ motor skills data were collected at follow-up visits, and parents or caregivers were interviewed for neonatal and neurodevelopment information. Assessments were administered and scored by trained child and adolescent neuropsychiatrists and by neuropsychomotor rehabilitation therapists.Cognitive assessment was conducted using standardized instruments, not yet specifically validated for NMDs, but universally recognized as reference points for developmental and cognitive assessment in developmental age. For children aged between 1 and 42 months, the Italian validated version of Bayley Scale of Child and Infant Development Third Edition was performed .Wechsler Preschool and Primary Scale of Intelligence, Third Edition (WPPSI-III), for children aged between 2 years and 6 months and 7 years and 3 months.Wechsler Intelligence Scale for Children, Fourth Edition (WISC-IV), for patients aged between 6 years and 16 years and 11 months. The Italian standardization of the Wechsler scales  was useThe Wechsler scales are standardized tests that yield an FSIQ, representing overall intellectual ability, and index scores that measure different domains such as verbal intelligence, non-verbal intelligence, working memory and processing speed. WPSSI-III sub-indices are VIQ, PIQ and processing speed quotient (PSQ), while WISC-IV sub-indices are verbal comprehension index (VCI), perceptual reasoning index (PRI), working memory index (WMI) and processing speed index (PSI).We used the Italian standardized normative data provided in the Wechsler IQ tests for comparison. Both the WISC-IV and WPPSI III have large normative data that are reported to be representative of the Italian speaking population and are stratified by age, gender, ethnicity, education level, and geographical region. n = 2200 subjects , aged between 6–16 years, 11 months, and 30 days, attending primary, middle and high-school.WISC-IV: n = 987 subjects, aged 2 years and 6 months–7 years and 3 months, attending public school and kindergarten, proportioned to the Italian general population.WPSSI-III: The details of normal population are as follows: The normative population mean across both measures is 100 with a standard deviation of 15 for sub-indices scores and have a mean of 10 with a standard deviation of 3 at the subtest level.Motor function assessment was performed using standardized rating scales, many of which have already been validated for NMDs, and are used in usual practice.All the scales are described and are summarized in Descriptive analyses were performed for all the demographic and clinical variables of interest .Concerning analyses on cognitive profile, all assessment instruments described above have been standardized for use in Italy and, therefore, the mean and SD for each subscale in a normative population is known. Consequently, for DMD, BMD, DM1 and HMSN subgroups, given that a difference with normal distribution could not be found with a Shapiro–Wilk test for FSIQ and sub-indices distribution, the one-sample Z-test was applied to compare FSIQ and sub-indices mean scores with normative data provided in the Italian validated version of Wechsler IQ scales. Descriptive results data were provided for SMA patients group.When appropriate, Pearson’s simple linear correlation analyses were used to investigate possible associations between motor function scores and cognitive performance within NMD subgroups. In both DMD and DM1 cohorts, the Fisher exact test was run to investigate a possible association between ID  and a delayed walking acquisition , and between the presence of ID and a delayed language development . t-test was used to evaluate the relationship between the location of the mutation in the DMD gene  and the WISC-IV and WPSSI-III FSIQ scores together. Moreover, in DM1 patient cohort, the t-test was performed to investigate possible FSIQ score differences on WISC-IV scale between the congenital and the infantile form. In the DMD sample, For statistical analyses, a two-tail significance level α of 0.05 was set; regarding FSIQ and sub-indices on Wechsler scales, comparative analyses with normative population data, a Bonferroni correction for multiple comparisons was applied, giving an α of 0.01 (five tests) for WISC-IV, and a α of 0.0125 WPSSI-III (four tests). Statistical processing was performed using the IBM SPSS Statistic software, version 24 .The sample included a total of 43 pediatric patients: 15 subjects affected by DMD, 4 by BMD, 8 by DM1, 6 by GSD2, 3 by type 2 or 3 SMA, and 7 by type 1 or 2 HMSN.Patients’ full demographics and clinical characteristics are summarized in Fifteen DMD patients, all male, with a median age at assessment of 6.7 years  were recruited.Twelve subjects (80%) had a wide out-of-frame deletion in the locus Xp21 dystrophin gene, one patient had an out-of-frame duplication of exon 44, and two patients (13%) had a point mutation within exon 43.n = 7), or WISC-IV (n = 8) based on the patient’s age. FSIQ was mean 96.9 (SD 26.1) on WPSSI-III scales, and 75.9 (SD 11.1) on WISC-IV scales, with no significant difference between the two FSIQ means (t-test p = 0.08). Eleven patients (73%) had an FSIQ score on WPPSI-III or WISC-IV scales greater than 70, in particular 4 (36%) were in the borderline range  and 2 (18%) in the high range  compared to normative data for age. Four subjects  had an FSIQ lower than 70, presenting an ID of mild degree. No case of moderate or severe ID  was found in this cohort. Cognitive function was assessed with WPPSI-III (p < 0.0001), while the WPPSI-III FSIQ mean score was not significantly different .Compared to the normative population FSIQ mean scores, WISC-IV FSIQ mean score was significantly lower   .p > 0.01).On the WPPSI-III scales, all the four sub-indices mean scores were not significantly different compared to normative mean scores , while no significant correlations were found between the 6-minute walking test (6MWT) total scores and WPPSI-III or WISC-IV FSIQ scores (A significant correlation between WISC-IV FSIQ scores and North Star Ambulatory Assessment (NSAA) total scores was found . Since in literature there is growing evidence that intellectual functioning in DMD patients is variable depending on whether the mutations are located proximally or distally within the Dys gene, with a greater negative impact for more distal mutations, association analyses were performed to investigate a possible difference in this cohort. The cut-off, to discriminate between proximal and distal, was established by the Authors prior to exon 30  or exon p = 0.08 in both cases).No significant associations emerged between a history of delayed language or walking acquisition and the presence of ID at assessments  were enrolled. All patients had in-frame deletions in the dystrophin gene. Two subjects had never presented neuromuscular symptoms, while in the other two the disease started with fatigue and cramps/rhabdomyolysis at 3 and 6 years of age respectively . p > 0.01 in all cases)  .Eight patients, 4 males, with a median age at assessment of 12.9 years  were assessed.n = 2) or WISC-IV scales (n = 6), based on the patient’s age.Cognitive assessment was performed with the Bayley-III , with ID in four out of six patients, of mild degree in one patient and moderate in the other three. Of the two patients with a FSIQ score greater than 70, in one case the FSIQ score was at the lower limits of the norm for age. All WISC-IV sub-indices mean scores were significantly lower compared to the normative population mean score . . .p < 0.05), while no significant correlation was found between 6MWT scores and WISC-IV FSIQ scores  scale , while no association emerged with a history of delayed walking acquisition.Moreover, in this cohort, a significant association emerged between a history of delayed language acquisition and the presence of ID at the study assessment  and the degree and frequency of cognitive impairment: a greater severity of cognitive impairment was found in the congenital form compared to the infantile one . Based on previous reports ,27, it wSix patients, 4 males, with a median age at assessment of 8.4 years  were enrolled. Two patients were affected by the classical infantile form of GSD2 disease, while 4 by the later onset form. At the time of assessment four patients were being treated with ERT, while two, both affected by the later onset form and asymptomatic, were not receiving ERT.° and 6° percentile rank respectively) in both, while remaining in the normality range for age. One patient had low scores for age in the motor area (2° percentile rank), with a greater involvement of gross motor skills, and in Adaptive Behavior Scale (2° percentile rank).The two patients assessed with the Bayley-III scales scored in the normal range for age on the cognitive scale, with an associated fall on the language scale   .Three SMA patients, two with type 2 and one with type 3, 2 males, with a median age at assessment of 10.7 years , were enrolled.Cognitive function was assessed by WISC-IV scales in all three subjects showed. FSIQ median score was 114.0 (IQR 5.0), and, in particular, FSIQ score was in the normal range in two patients and in the higher range compared to population data for age in the remaining one . Concerning WISC-IV sub-indices scores, for all three patients the PSI score was in the normal range, with a median of 97.0 (IQR 1.5), while the VCI score was a point of strength, with a median of 118.0 (IQR 1.0). In one patient also the PRI was a strength (PRI index score 119).Five patients, 2 males, with a median age at assessment of 11.5 years , were enrolled. Two patients were affected by type 1A form, two patients (brothers) by type 1B, while one patient was affected by type 2A form . n = 1) and WISC-IV scales (n = 6). The WISC-IV FSIQ mean score was 101.7 (SD 6.2), with all subjects having a FSIQ score in the normal range. FISQ and sub-indices mean scores were not significantly differentce compared to normative data   . Individual scores for each NMD are reported in In this study, the global intellectual functioning of children with NMDs of different etiology was systematically assessed and compared with the Italian normative population data for age provided by Wechsler IQ scales. Furthermore, to investigate possible associations with the degree of motor impairment, in the whole sample the motor function was systematically measured through validated motor performance tests. Because of the low population prevalence of NMDs, the study sample size was rather small for precise statistical estimates, and there was numerical heterogeneity among NMD subgroups, patients age and, therefore, among cognitive and motor function assessment scales used. However, despite these limitations, several inferences can be drawn. In general, it is not easy to obtain a standardized assessment of intelligence in children with neuromuscular disabilities, especially when severe. In fact, many of the standard cognitive tests for older children are based on general knowledge and experience, which can be limited precisely because of motor disabilities. Furthermore, many tests depend on motor ability and speed of manipulation and coordination, abilities that can be reduced by muscle weakness. However, despite these challenges, the administration of the cognitive assessment scales proved overall to be feasible in the study sample.The results indicate that cognitive abilities in developmental age seems to be quantitatively and qualitatively affected in children with NMDs. Consistently with previous reports ,41, in tn = 3) and clinical heterogeneity prevented statistical testing. Despite the important limitation, this finding is in general consistent with previous reports that found FSIQ scores in the normal range or above in SMA [On the other hand, in our sample, on the Wechsler IQ scales , FSIQ and its sub-indices mean scores were not significantly different from the normative mean score in DMD patients below 6 years of age and in BMD, GSD2, HMSN and type 2 or 3 SMA subgroups, which is consistently with the literature . In the e in SMA .It should be highlighted that some tests that contribute to PIQ and PSI of the Wechsler IQ scales, such as symbol search and coding, may be influenced by motor speed and, therefore, muscle function ; howevern = 2, GSD2 n = 2), on the Cognitive Scale DM1 patients scored lower than expected for age, while GSD2 patients scored in the normal range, consistent with previous literature report [Of the four children assessed by Bayley-III scales (DM1 e report . On the Nonetheless, the systematic cognitive assessment design for this study highlighted the presence of early delays, namely in DM1, underlying once again the importance of conducting an early developmental assessment in children with NMDs to identify possible areas of intervention that may improve clinical management and quality of life of patients and their caregivers.In the DM1 subgroup, a history of delayed language acquisition was associated with the presence of an ID at the time of the assessment. This finding may suggest that an early delay in language, even before the onset of significant muscle weakness, cannot be exclusively attributed to muscle impairment, emotional reaction to illness, or lack of experience opportunities related to motor limitations and, therefore, must prompt cognitive assessments and follow-up in time.When considering the genotype–phenotype relationship in the sample, no differences in FSIQ scores emerged between proximal and distal dystrophin mutations in the DMD subgroup, but the small sample size provided only limited statistical power.In the study sample, consistently with previous reports, a greater severity of cognitive impairment was found in the congenital form compared to the infantile one in the DM1 subgroup .Considering the possible associations between cognitive and motor functions, this study found that in the DM1 subgroup, lower WISC-IV FSIQ was associated with lower scores on the motor function assessment scale MFM32, and that, in the DMD cohort aged more than 6 years, lower WISC-FSIQ scores were associated with lower NSAA scores. It is noteworthy, however, that these associations, do not allow cause to be discriminated from effect. It is challenging to establish the direction of the relationship between motor and cognitive function, i.e., whether motor deficits negatively affect the cognitive development of patients or, vice versa, it is the cognitive deficit that limits motor performance. It is likely that in DM1 there is a common pathogenetic mechanism of damage to the neuromuscular system and the CNS.No significant associations between WISC-IV or WPSSI-III FSIQ and total scores on the motor function scales were found in the DMD cohort aged under 6 years and in the BMD, GSD2, SMA and HMSN subgroups. This result may be again due to the small sample size.As already acknowledged, the main limitation of this pilot study is the small sample size and a very small number of patients in the NMD subgroups. A second major limitation is the absence of an internal control group. A third limitation is the heterogeneity in disorders age and developmental stage requiring the utilization of different motor and cognitive scales; this heterogeneity constitutes a major limitation that prevents generalizations about the relationship between motor deficit and intellectual functioning in NMDs. A further limitation is that some tests that contribute to PIQ and PSI on the Wechsler scales, such as symbol search and coding, may be influenced by motor speed. Moreover, in various NMD diseases, patients may already show at early developmental age an impairment in vital functions, thus further complicating the heterogeneity of the population and the results. Finally, the lack of neuroradiological data prevents the identification of possible structural brain abnormalities. However, brain MRI examinations are not routinely conducted without specific clinical indications, also because they require pharmacological sedation in young children.This study adds to previous findings of several cognitive phenotypes in children and adolescents with NMDs and provides evidence of the feasibility and the importance of a regular and early assessment of cognitive functions in these patients, in order to enable interventions aimed at preserving functioning and quality of life. This seems especially relevant and timely, in the light of the increased survival brought by the recently introduced innovative therapies.Moreover, a specific focus on cognitive deficits in these disorders can help to reduce the global pathology-related family burden.Finally, the availability of new therapies, namely in SMA and GSD2, paves the way unexpected developments. This finding should prompt the inclusion of cognitive assessment in the follow-up protocols at visit of specialized tertiary neuromuscular centers.Further studies with larger samples are needed to characterize trajectories of cognitive, neuropsychological, and adaptive functioning, with a special attention to new phenotypes that are emerging with the availability of innovative therapies.


Actinidia eriantha) is a peculiar berry resource in China, and the maturation period is generally late. Fortunately, we found an early mature A. eriantha germplasm. In order to explore the formation mechanism of its early mature trait, we determined the main carbohydrate and endogenous hormone content of the fruit, and used off-target metabolomics and transcriptomics to identify key regulatory metabolites and genes. We found that early mature germplasm had faster starch conversion rate and higher sucrose, glucose, and fructose content when harvested, while with lower auxin (IAA), abscisic acid (ABA), and zeatin (ZR) content. Through the non-targeted metabolome, 19 and 20 metabolites closely related to fruit maturity and early maturity were identified, respectively. At the same time, weighted correlation network analysis (WGCNA) showed that these metabolites were regulated by 73 and 99 genes, respectively, especially genes related to sugar metabolism were mostly. Based on above, the formation of early mature trait of A. eriantha was mainly due to the sucrose decomposition rate was reduced and the soluble solid content (SSC) accumulated at low levels of endogenous hormones, so as to reach the harvest standard earlier than the late mature germplasm. Finally, ten single nucleotide polymorphism (SNP) loci were developed which can be used for the identification of early mature trait of A. eriantha.Kiwifruit ( Actinidia eriantha) has been studied for the first time.- The formation mechanism of early mature trait of kiwifruit (Actinidia eriantha) is a unique germplasm resource in China and widely distributed in the Yangtze River basin, the Yunnan–Guizhou plateau, and the Sichuan basin, at altitudes 200–2,000 m  during the investigation of wild kiwifruit resources , insert or delete maker (InDel), structural variation (SV), and copy number variation (CNV) loci of key regulatory genes, and develop SNP loci that can be used for early mature germplasm identification. This study is expected to reveal the physiological basis and molecular mechanism of the formation of early mature trait of A. eriantha germplasm, ‘Ganlv 2’, and late mature germplasm, ‘Ganlv 1’, were selected as the experimental materials, which planted in Shanwei kiwifruit germplasm resource nursery  in Fengxin County, Jiangxi Province, PR China. The parent materials of ‘Ganlv 1’ and ‘Ganlv 2’ were collected in the same place, had similar botanical traits and flowering stages, and the genetic similarity coefficient value was less than 0.08  of 6.5% is the general standard for fruit harvested of A. eriantha.According to our previous method , we conda content (Chl a), chlorophyll b content (Chl b), carotenoids content (Car), ascorbic acid content (AsA), starch content, and sugar components. Except for starch content was determined by iodine colorimetry , soluble sugar content (TS), titratable acid content (TA), chlorophyll orimetry , other iorimetry . In addiorimetry , kiwifruA. eriantha, we commissioned Genedenovo Biotechnology Co., Ltd  to perform non-targeted metabolomics sequencing. The extraction, sequencing, and bioinformatics analysis of metabolites were carried out according to routine procedures. LC-MS/MS analyses were performed using an UHPLC system  with a UPLC BEH Amide column coupled to QE HFX mass spectrometer . The QE HFX mass spectrometer was used for its ability to acquire MS/MS spectra on information-dependent acquisition (IDA) mode in the control of the acquisition software . The raw data were converted to the mzXML format using ProteoWizard and processed with an in-house program, which was developed using R and based on XCMS, for peak detection, extraction, alignment, and integration. Then, an in-house MS2 database (BiotreeDB) was applied in metabolite annotation. Discriminant orthogonal partial least squares discriminant analysis (OPLS-DA) was used for model testing and screening of differential metabolites. This method can effectively reduce the complexity of the model and enhance the explanatory ability of the model without reducing the predictive ability of the model, so as to maximize the difference between groups.In order to more comprehensively analyze the physiological basis of the formation of early maturity trait of A. eriantha ‘White’ genome was as reference genome for data analysis  according to the protocol of the manufacturer. The quality of RNA was detected by an ultra-differential photometer and after, the mRNA with polyA tail was enriched with magnetic beads with OligodT. The obtained RNA was fragmented with the interrupted buffer, reverse transcription with random N6 primer, and then synthesized the cDNA two-strand to form double-stranded DNA. Then, the synthetic double-stranded DNA was filled in and the 5′ was phosphorylated. The 3′ forms a sticky end with an ‘A’ protruding, and then a blister linker with a protruding ‘T’ at the 3′ was connected. The ligation product was amplified by PCR with specific primers. The PCR product was heat-denatured into single-stranded, and then the single-stranded DNA was circularized with a bridge primer to obtain a single-stranded circular DNA library. Finally, it sequenced by BGISEQ-500 by BGI Medical Laboratory Co., Ltd . analysis . SequencA. eriantha ‘White’ genome was also used as reference genome for data analysis. The sequencing process was carried out according to the conventional process. After filtering the raw data obtained by the instrument, GATK software was used to obtain SNP and InDel information, Breakdancer software to detect SV sites  analysis.The experimental data were statistically analyzed and processed with Microsoft Excel 2016 and SPSS statistical package (IBM SPSS Statistics 22.0). A two-tailed Student’s According to previous research ideas , we seleAccording to the distinctness, uniformity, and stability (DUS) test results, it can be seen that the early maturity trait of ‘Ganlv 2’ can be stably inherited, and the phenological period of the early maturity cultivar ‘Ganlv 2’ and the late maturity cultivar ‘Ganlv 1’ is only different in fruit maturing time, the fruit of ‘Ganlv 2’ maturated in early October, and the fruit of ‘Ganlv 1’ maturated in late October, about 20 days later than that of ‘Ganlv 2’ .a, Chl b, and Car of ‘Ganlv 1’ and ‘Ganlv 2’ were consistent throughout the growth period, the contents of Chl a, Chl b, and Car of ‘Ganlv 1’ were significantly higher than that of ‘Ganlv 2’ at S1–S3 period, but lower than that of ‘Ganlv 2’ at S7  and negative ion mode (NEG) to detect metabolites. There were differences in the total number of metabolites identified in the two ionization modes. POS and NEG modes identified 4,658 and 2,806 metabolites, respectively. Among them, the known metabolites were 1,378 and 440, and most of them were unknown metabolites.OPLS-DA can effectively reduce the complexity of the model and enhance the explanatory ability of the model without reducing the predictive ability of the model to maximize the difference between groups. As can be seen from In order to verify the reliability of the OPLS-DA model, cross-validation and permutation tests were carried out on the model. The cross-validation results showed that no matter in the POS mode or the NEG mode, the reliability of the models of all comparison groups was high, the model predictive ability was good, and met requirements of the analysis . The perIt can be seen from A. eriantha . The results showed that 2,433 genes related to maturity can be divided into three color modules, namely turquoise module , blue module (561), and gray module (13) . At the In terms of genes and metabolites related to the formation of early mature trait, WGCNA analysis results showed that 3,132 genes related to the formation of early mature trait can be divided into four color modules, namely, the brown module (931), the turquoise module , blue modules , and gray modules (3) . A totalThe relative expression of 26 key candidate genes was detected by qRT-PCR, and the quality of transcriptome sequencing was detected by correlation analysis. As can be seen from A total of 45.76 G of original sequencing data was generated by sequencing, and 44.65 G of high-quality and usable data was filtered by SOAPnuke software. The Q20 of ‘Ganlv 2’ and ‘Ganlv 1’ were both greater than 95%, Q30 was greater than 90%, the sequencing depth was 28.95 X and 32.46 X, respectively, and the matching ratio of the whole genome to Wat was 98.87% and 98.72%, respectively. The mapping rate to the whole genome of ‘white’ was 98.87% and 98.72%, respectively.Compared with the reference genome, ‘Ganlv 2’ and ‘Ganlv 1’ had 9,399,983 and 9,487,607 SNP sites, respectively. Among them, 9,393,020 and 9,306,154 were homozygous SNP sites, 5,839,927 and 5,785,585 mutation sites were converted type, and 3,622,964 and 3,588,924 mutation sites were transverted type.There were 3,574,882 and 3,538,599 InDel sites on ‘Ganlv 2’ and ‘Ganlv 1’, respectively. Among them, 3,400,627 and 3,365,812 were homozygous InDel sites, 174,255 and 174,255 were heterozygous InDel sites, 1,841,505 and 1,822,968 InDel sites were insertion, and 1,733,377 and 1,715,631 InDel sites were deletion.The insertion SV sites of ‘Ganlv 2’ and ‘Ganlv 1’ were 0, and the deletion SV sites were 16,506 and 5,405, respectively. Inversion SV sites were 17,705 and 10,970, respectively. There were 17,071 and 14,714 intra-chromosomal translocation SV loci, and 48,184 and 29,906 inter-chromosomal translocation SV sites, respectively.The loss number and gain number of CNV on ‘Ganlv 2’ was more than that of ‘Ganlv 1’. They were 340 and 256, 190 and 62, respectively.Finally, Circos software was used to draw the information of various mutation sites on chromosomes on a map . In addiWe use the SnpEff software to classify the SNP sites on the early maturity germplasm ‘Ganlv 2’ according to the degree of influence of SNP on the protein structure. About 0.061%  of the SNP sites have a serious impact on the protein. According to the influence of the mutation site on protein coding, the mutation sites were classified. In order to ensure the accuracy of SNP sites and filter out low-quality sites, the sequencing depth was required to be ≥10X. There was a total of 42,969 genes with differential SNP sites on ‘Ganlv 2’, of which 7,331 SNP sites were key sites that seriously affect the structure of the protein. Combined with the analysis results of the identification of genes related to the formation of early mature trait, there were 24 SNP sites distributed on seventeen key candidate genes related to the formation of early mature trait . After fA. eriantha , and fruit ripening  (EC: 2.4.1.13) and sucrose phosphate synthase (SPS) (EC: 2.4.1.14) were mined out through transcriptome technology. Both are key enzymes involved in the process of sucrose synthesis and accumulation, and are mainly distributed in the cytoplasm. Among them, SUS is the only enzyme that enables sucrose to participate in various metabolic processes such as tissue construction, material storage, and plant cell metabolism, and it exists in most plants in the form of more than two isoenzymes . HoweverA. eriantha is mainly due to the significant metabolic differences in sugar metabolism. At S7 stage, the conversion efficiency of sucrose to fructose and glucose in the early mature germplasm fruit was reduced, the SSC was maintained and the rapid rising trend appeared so as to reach the harvested standard earlier than the late mature germplasm. As the formation of early mature trait, the gluconic acid and carnosic acid in the organic acid components were significantly reduced and accompanied by a large accumulation of volatile aroma substances precursor synthetic substances, which ultimately leads to the formation of the early mature trait of A. eriantha  can be found in the article/GL: data analysis and writing-original draft preparation. YH and YL: collect fruit materials and fruit quality determination. QL and HW: metabolome data analysis. BY and DJ: real-time fluorescence quantification. MZ and CH: transcriptome data analysis. XX: writing-reviewing and editing. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The COVID-19 pandemic is a public health emergency of international concern, and the main measures to contain the spread of the coronavirus causing COVID-19 were social distancing, quarantine, and self-isolation. Although these policies are effective in containing the spread of the virus, they might represent a challenge to psychological well-being, increasing levels of depressive and anxiety-related symptoms. Aims: We explored the frequency of anxiety and depression symptoms during COVID-19 restrictions and associations with sociodemographic factors in a Brazilian sample. Method: Data of a total of 936 Brazilian adults (68.2% women) aged 18 to 77 years old  were collected through an online survey. Results: In general, we observed a frequency of 17.36% for severe anxiety and 66.13% for severe depression symptoms, in which younger participants (18–39 years old) and women showed higher scores in anxiety and depression scales compared to older age groups. Logistic regressions showed that women were more likely to present severe symptoms of anxiety (20.4%) compared to men (10.9%), as well as respondents in the educational sector (24.3%) compared to those in the health sector (10%). Conclusions: We highlight the importance of mental health professionals in developing strategies to help younger adults to mitigate the effects of social restriction. The COVID-19 pandemic is a public health emergency of national and international interest. Confinement measures represent the best way to prevent the spread of the virus, and yet, a challenge to psychological well-being , the posIn fact, the results of studies assessing the immediate psychological impacts and associated factors during the COVID-19 outbreak among China’s general population showed that 28.8% of respondents had rates corresponding to moderate to severe anxiety symptoms, and 6.5% reported moderate to severe depressive symptoms. Greater psychological effects of the pandemic were associated with being a woman or/and a student . Similarly, studies performed in Europe revealed an increased prevalence of anxiety and depression in younger populations, specifically in Spain , Italy ,13, and Although more developed countries have relief funds to support their citizens during the pandemic, another essential finding was, for instance, in China, that a protective factor was the stability of family income, since those participants without a fixed family income showed increased anxiety , which mAccording to the last World Health Organization  report, Beyond the inherent socioeconomic inequalities aggravated by the measures of confinement, since the beginning of the COVID-19 outbreak, Brazil’s public health messages on the severity of the virus were implemented slowly and with lower strictness than in other countries , and “faIndeed, a study carried out in Brazil by Campos et al.  with 12,Another study performed by Serafim et al. , also inFinally, Feder et al. , throughAlthough we observed published studies, including Brazilian samples exploring the frequency of anxiety and depression, studies examining whether participants were tested for COVID-19 and those looking at economic changes during social restriction measures and their impact on mental health are scarce.For this reason, we sought to evaluate the frequency of anxiety and depression symptoms in a Brazilian sample; to assess mental health states in different age groups and whether participants with changes in their income would present differences in depression and anxiety symptoms to those whose income did not change due to social restriction measures. Furthermore, we aimed to observe how many participants were tested for COVID-19. Finally, we investigated possible factors influencing severe anxiety and depression symptoms during the COVID-19 containment measures in the spring of 2020. We hypothesized that the frequency of anxiety and depression symptoms would be high due to misinformation regarding the pandemic . MoreoveThis study used data from two surveys carried out in Brazil that have received ethical approval by the responsible Ethics Committees, which were São Paulo State University “Júlio de Mesquita Filho” Bauru Campus (Process: 4.021.098) and Gaffrée and Guinle University Hospital, Rio de Janeiro (Process: 4.125.060). We obtained electronic informed consent from all participants before they began to fill in the online survey. We estimated the sample size using Raosoft software, using a conservative assumption, we applied a level of confidence of 95%, with a 50% response distribution and an error margin of 5%. The minimum sample size recommended was 385 participants.n = 24 respondents. Thus, a total of 936 participants who completed at least 90% of the survey were included in the analysis. All respondents lived in Brazil, with 86.15% in the southeast states, 5.87% in the northeast, 4.66% in the south, 1.88% in the north, and 1.44% in the center west.Participants of both studies answered the questionnaires and scales anonymously through an online survey running from 27 April to 5 July 2020, and they received no compensation for it. We applied a snowball sampling approach since it was not possible to establish the sample’s randomness due to the need of starting data collection as soon as possible. In total, 981 respondents started the survey; however, 8 of them were under 18 years old and were excluded from the final analysis. Only participants who reported living in a city with social distance restrictions in place were included in this study, which led to the exclusion of We used two different surveys, one released in São Paulo and the other in the Rio de Janeiro state. However, the questionnaire items analyzed in this study were collected with similar wording, and the depression and anxiety scales were the same. Aiming to unify the datasets with varied formats, we harmonized the studies of both data, transforming it into one cohesive dataset with the variables of interest for this study. The online survey was available through the Google Forms platform and comprised the three sections described below.This questionnaire contained questions on age, marital status, gender, living arrangements, nationality, state, formal education, professional sector, and socioeconomic factors such as monthly income.This questionnaire contained questions adapted from the World Health Organisation guidelines ; for insTwo mental health scales were included to assess anxiety and depression. The Generalized Anxiety Disorder (GAD-7)  assessesParticipants were recruited through social media . Those who clicked on the link provided in the posts were redirected to the questionnaire hosted on Google Forms. Firstly, respondents read the informed consent and agreed to participate by clicking “I agree to participate”. An e-mail address created for this study was available to participants if they had any questions or concerns, but no events were reported. Then, participants were asked to fill in the three sections of the survey consecutively. Participants took about 20–30 min to finalize the survey.2). Second, we confirmed the normality of continuous variables and carried out t-tests and one-way ANOVAs with Bonferroni for multiple comparison post-hoc tests to analyze the differences between sociodemographic characteristics in anxiety and depressive symptoms. Third, we performed χ2 tests to compare possible differences among age groups and sociodemographic characteristics. Finally, we performed two separated multiple logistic regression models to investigate whether sociodemographic or COVID-19 related variables would influence severe anxiety and depression classifications during the COVID-19 pandemic. To select the variables that would be included in these models, simple logistic regressions were carried out with each sociodemographic and COVID-19-related variable. To provide a full account of potential covariates of anxiety and depression, all variables with significance levels equal to p < 0.05 were included in the multiple logistic regression models. We reported the odds ratio (OR), 95% confidence interval (95% CI), and p values. All statistical analyses above cited were conducted through STATA  software.First, descriptive analyses were conducted to describe the demographic characteristics of the sample and the frequency of social distancing behavior , and changes in socioeconomic factors  using Chi-square tests (χThe demographic characteristics of our sample are displayed in n = 42) of the sample reported that they had been tested for COVID-19. Regarding the pandemic’s socioeconomic impact, 15.93% (n = 147), respondents were not receiving their salaries due to COVID-19 pandemic restrictions. Those respondents employed in the education sector reported leaving the house fewer times per day (37.7%) compared to other professional sectors , χ2 = 24.97, p < 0.001.A total of 4.54%  were more likely to present changes in their salaries due to COVID-19 measures compared to post-graduates (10.51%), χt-tests, with sex as the independent variable and depression and anxiety symptoms as dependent variables, revealed that women reported higher scores for both scales  compared to men , t (930) = 4.04, p < 0.001, t (930) = 4.90, p < 0.001, respectively. Regarding the t-tests for changes in income due to COVID-19 and depression and anxiety symptoms, we observed no differences, t (931) = −1.79, p = 0.07, t (931) = −0.32, p = 0.75, respectively.The analyses performed with ps < 0.005. The ANOVAs results are displayed in ps < 0.001). Individuals from the professional education sector revealed higher depression symptoms in contrast with unemployed, health and other sectors (ps < 0.001). Respondents who reported they were “working at the moment” showed lower depression scores than those not working (p = 0.002). In the following, we report group differences and bivariate associations with 2 tests showed that younger participants (67.63%) were more likely to have post-secondary education than the other age groups (χ2 (8) = 115.78, p < 0.001). Younger people were more likely to be single, at 85.56%, χ2 (8) = 115.78, p < 0.001. However, the young age group was more likely to live with their parents (63.54%) when compared to the other age groups (36.46%), χ2 (12) = 257.33, p < 0.001. Furthermore, younger groups were more likely to receive 1–3 wages compared to the other age groups, χ2 (12) = 86.61, p < 0.001, as well as to be unemployed (14.39%) compared to other age groups, χ2 (12) = 74.14, p < 0.001. Finally, young people were less likely to be working in comparison to those who were 30–59 years old, χ2 (4) = 60.80, p < 0.001.Moreover, χ2 (3) = 18.01, p < 0.001. In addition, more women reported working in a health profession (25.39%) compared to men (13.13%), χ2 (3) = 30.28, p < 0.001.Regarding sex, 41.22% of women reported not leaving home compared to 27.37% of men, χIn As shown in The main goal of this study was to evaluate the frequency of anxiety and depression symptoms of a Brazilian sample during COVID-19 confinement measures and explore whether socioeconomic factors would be associated with symptoms of anxiety and depression. As predicted, our findings indicated a high frequency of anxiety symptoms and a very high frequency of depression affecting most of the Brazilian sample during the COVID-19 pandemic. These results are consistent with studies assessing mental health in the Brazilian population ,28,29,31An alarming finding of this study was that only a small minority of the sample (4.54%) was tested for COVID-19. This result unveils underestimated numbers of cases and corroborates that Brazilian testing rates are among the lowest in the world, with approximately 3462 people tested per 1 million inhabitants during the time of data collection . This unAlthough older people are the primary mortal victims of the COVID-19 pandemic, our results suggested that the mental health of young people is highly impacted by a scenario that combines low income, lack of autonomy and reduced social interaction ,29,31,40Moreover, women presented higher prevalence of anxiety and depression symptoms, which is also congruent with studies performed in Brazil ,29,30,31The prevalence of severe depressive symptoms was higher compared to studies running during the COVID-19 pandemic in high-income countries ,12,13,14Our study detected a prevalence of 17.36 % of severe anxiety symptoms, which, compared to two studies carried out in China, was lower than the anxiety rate of 35% ,45 but cAs governmental directives were ambiguous, we observed that only 36.79% of the respondents were following social distancing measures, which explained the exponential spread of the virus, regardless of the new variant. In some countries, government attitudes and campaigns led to a sense of a collective identity in which individual actions account for the protection of the entire society . That isBy contrast, educational professionals presented higher levels of anxiety and depressive symptoms compared to professionals in the health sector. This result may be due to the uncertainty and stress among teachers because of the abrupt closure of schools and the need to adapt to new working conditions, such as distance education . MoreoveThe strength of our study was a timely data collection and a large sample of respondents from the most populous regions of Brazil , which iIt would have been ideal to have in-person psychiatric assessments for the clinical diagnosis of anxiety and depression. However, pandemic restrictions did not allow for such a procedure at the time. To partly compensate for this shortcoming, we only chose validated scales for the assessment of anxiety and depression.One of the limitations of the study is a lack of pre-pandemic levels of depression and anxiety for the sample. Furthermore, the randomization of our sample was not possible due to the use of a convenience online survey to prevent infections, which could cause sample bias, and which resulted in a sample that was not balanced in terms of residence across states of Brazil. In fact, we have to recognize that our sample was highly educated and in better socioeconomic standing than a population-representative sample. Even though some participants had their economic situation affected, they were in the minority, and likely underrepresented in our sample. Despite earlier evidence that showed higher anxiety prevalence, particularly for those with lower socioeconomic standing ,16, we fFinally, the results of this study reinforce the importance of paying special attention to young adults, since, for those affected by mental health disorders, it may go along with other negative unintended and long-term harmful consequences, such as a decreased ability to work and increased healthcare costs. We suggest that younger adults could benefit from mental health care through psychoeducation programs such as resilience training. For example, coping strategies can promote behaviors that protect against the negative symptoms caused by the necessity to follow social distancing rules ,57.In conclusion, the findings underline the importance of mental health clinicians and researchers in developing strategies to mitigate the psychological impact of restrictive pandemic control measures, mainly on young adults. Providing additional psychological support measures should be a public health priority during and in the aftermath of the COVID-19 pandemic.


A non-invasive neurorehabilitation multimodal protocol (NRMP) may be applicable to chronic T3-L3 dogs 3 months after undergoing surgery for acute Intervertebral Disc Disease (IVDD) Hansen type I; this protocol has been shown to be safe, feasible, and potentially effective at improving ambulation in both open field score (OFS) 0 and OFS 1 dogs. The specific sample population criteria limit the number of dogs included, mainly due to owners withdrawing over time. Thus, the present case series study aimed to demonstrate that an NRMP could contribute to a functional treatment possibly based on synaptic and anatomic reorganization of the spinal cord.n = 9) and OFS 1/DPP+ (n = 7). All were enrolled in the NRMP for a maximum of 90 days and were clinically discharged after achieving ambulation. The NRMP was based on locomotor training, functional electrical stimulation, transcutaneous electrical spinal cord stimulation, and 4-aminopyridine (4-AP) pharmacological management. In the Deep Pain Perception (DPP)+ dogs, 100% recovered ambulation within a mean period of 47 days, reaching OFS ≥11, which suggests that a longer period of time is needed for recovery. At follow-up, all dogs presented a positive evolution with voluntary micturition. Of the DPP− dogs admitted, all achieved a flexion/extension locomotor pattern within 30 days, and after starting the 4-AP, two dogs were discharged at outcome day 45, with 78% obtaining Spinal Reflex Locomotion (SRL) and automatic micturition within a mean period of 62 days. At follow-up, all dogs maintained their neurological status. After the NRMP, ambulatory status was achieved in 88% (14/16) of dogs, without concurrent events. Thus, an NRMP may be an important therapeutic option to reduce the need for euthanasia in the clinical setting.This case series study aimed to evaluate the safety, feasibility, and positive outcome of the neurorehabilitation multimodal protocol (NRMP) in 16 chronic post-surgical IVDD Hansen type I dogs, with OFS 0/DPP− ( Spinal cord injury (SCI) is a commonly observed event in dogs following spinal trauma and Intervertebral Disc Disease (IVDD). Clinically, the SCI can be considered complete/incomplete if Deep Pain Perception (DPP) is absent (DPP−) or incomplete if DPP is present (DPP+) ,2.After IVDD Hansen type I, the spinal cord is able to sustain variable degrees of contusion and compression, depending on the extrusion velocity and amount of extruded disc material . DPP− doMore severely affected dogs can benefit from controlled activity programs in the field of neurorehabilitation. Functional neurorehabilitation belongs to physical medicine and rehabilitation in the area of restorative neurology and can be implemented in veterinary medicine , based oFunctional neurorehabilitation (FNR) protocols are based on repetitive task-specific therapy to optimize the spinal locomotor performance ,16,17 anDogs with chronic neurologic diseases can benefit from physical and rehabilitation support, which is common in human medicine, as well as in dogs and cats . After SIntensive FNR protocols modify motor interneuron intrinsic circuitry excitability and CPG through the stimulation of the cutaneous afferent pathway ,24 and tQuadrupedal step locomotor training could promote the activation of some long ascending and descending propriospinal interneurons ,25 and iPelvic limb muscle strength in SCI individuals can be improved by functional electrical stimulation (FES). FES includes efferent and afferent nerve stimulation, neuromuscular and muscle stimulation ,36, and,Intralimb hip–knee coordination may be promoted by the combination of FES and locomotor training ,38,39. IThere have been investigations regarding pharmacological management with 4-AP in thoracolumbar chronic SCI dogs to improve neurological deficits ,52,53. TChronic dogs are usually not modulated considering peripheral spinal reflexes, manifesting a non-coordinated and non-synchronized flexion/extension locomotor pattern with clonic reflexes. It is believed that 4-AP administration may enhance motor neuron pool excitability in dogs with depressed activity; however, studies with larger cohorts are needed in order to determine how this drug could be useful .Today, it is well known that the Central Nervous System (CNS) contains a complex network of neural connections, which are supported and modified by a population of glial cells, including astrocytes, oligodendrocytes, and microglia, which play an important role in neural plasticity. The astrocytes are the major components of the fibroglial scar that develops in the injury site after SCI . This glFurthermore, the fibroglial scar, which contains a rich mixture of cells and extracellular matrix , also inDespite this disparity regarding the glial scar, there is increasing evidence that suggests that it can support CNS repair, essentially via astrocytes, NG2 glia, and microglia.Historically, synaptic plasticity in pre-existent pathways and the formation of new circuits—in other words, synaptic and anatomical neuroplasticity, respectively—can promote the sprouting of lesioned fibers, contributing to regeneration . Thus, tThis case series clinical study intends to verify whether a neurorehabilitation multimodal protocol (NRMP) is a safe and feasible therapeutic protocol to be applied in SCI chronic dogs and to assess whether this NRMP allows a positive neurological evolution until ambulation and within a time period that is clinically acceptable.This study was conducted between March 2011 and January 2021 at Arrábida Veterinary Hospital and Rehabilitation Centre, (HVA/CRAA) after obtaining approval from the Lisbon Veterinary Medicine Faculty Ethics Committee and after obtaining the owners’ consent.Participants were included if they were chronic post-surgical IVDD Hansen type I dogs and more than 3 months had passed since their surgery. All had to be of chondrodystrophic breed, aged less than 7 years old, with a weight of less than 15 kg, with neuro-location at T10-L3, and classified at admission as paraplegic with DPP (OFS 1/ DPP+) or without DPP (OFS 0/DPP−). All DPP− dogs with voluntary movement of the tail, pain perception on the perineal region, scrotum, or vulva, and pain perception at the tip and base of the tail were excluded.The dogs were evaluated and the findings were documented (Canon EOS Rebel T6 1300D camera) by two Certified Canine Rehabilitation Practitioners (CCRPs) from the University of Tennessee and a non-CCRP neurologist from Lisbon University.The breed distribution was diverse: five (31%) Dachshunds, five (31%) French bulldogs, two (13%) Shi Tzus, two (13%) Pugs, one (6%) Pinscher, and one (6%) Pekingese. They had a mean age of 3.63 years (median of 4) and a mean weight of 7 kg (median of 7), and 56% (9/16) were males and 44% (7/16) were females. The most frequent neuro-location was L1–L2, for 37% (6/16) of dogs. Nine dogs were DPP− (56%) and seven dogs were DPP+ (44%).The dogs underwent a Home Work Plan (HWP) after the hemilaminectomy for Hansen type I IVDD, for 3 months, and performed three different types of protocols as described in All were enrolled in the NRMP for a maximum of 90 days and were clinically discharged after achieving ambulation. Ambulation was considered when dogs showed “the ability to rise and take at least 10 consecutive weight-bearing steps unassisted without falling” . RegardiAfter admission, the dogs were redirected to a neurorehabilitation consultation and evaluated based on their history and neurorehabilitation examination .For DPP evaluation, 12 cm Halsted forceps were used to test the response to a noxious stimulus applied to the bone of both medial/lateral digits of the pelvic limbs, always in a controlled environment, regarding movement and noise. Moreover, to perform an accurate pain examination, we also tested the perineal region and the tip and base of the tail.The flexion/extension locomotor pattern that was also monitored comprises interneurons interposed between the input and output. Thus, these interneurons allow divergence and promote input distribution to a wide population of output neurons. One type of input can then stimulate both agonist muscle contraction and antagonist muscle relaxation . In SCI,The dogs were then subjected to an NRMP. For DPP+ dogs, the protocol comprised a combination of locomotor training and electrical stimulation. Electrical stimulation was based on Functional Electrical Stimulation (FES) and Transcutaneous Electrical Spinal Cord Stimulation (TESCS). For DPP− dogs, the protocol implemented was the same as described, with the inclusion of pharmacological management with 4-aminopyridine (4-AP).This pharmacological management was applied in DPP− dogs because they were considered to have a complete SCI with a poor prognosis.Locomotor trainingThe locomotor training began on the second day of admission after the dogs had become accustomed to the land treadmill. Dogs were adapted with the help of technicians in a calm environment.The primary type of exercise was quadrupedal step training. For each training session, variables such as the walking speed and duration were increased and recorded, starting with 0.8 km/h (0.22 m/s) with a maximum of 1.9 km/h (0.53 m/s) ,64,65 foThe underwater treadmill (UWTM) training was introduced in all dogs from the second day of admission, with a water temperature of 26 °C  for 5 miLocomotor training was always associated with active and assisted kinesiotherapy exercises, such as postural standing, flexor movements, bicycle movements, balance board, and different floor surfaces  Table 4Table 4.Electrical StimulationThe electrical stimulation protocol applied in all dogs was a combination of FES, aiming to achieve muscle contraction and neural connection, and TESCS to increase the descending pathway depolarization.Functional Electrical StimulationA neuromodulation modality uses a short electrical pulse sequence to stimulate the lower motoneurons near the motor point region or by peripheric afferent stimulation, resulting in peripheral spinal reflexes ,77. ThisThe current parameters were as follows: 60 Hz and 6–24 mA ,79,80,81Transcutaneous electrical spinal cord stimulationThis is a non-invasive and non-painful rehabilitation therapeutic modality with the ability to neuromodulate the spinal cord’s physiological status .All dogs underwent TESCS three times a day (5 days/week), which was gradually discontinued when the flexion/extension locomotor pattern appeared; thus, the surface electrodes were placed in the paravertebral muscles (one electrode on L1-L2 and the other on L7-S1) and a continuous current of 50 Hz and 2 mA was applied for 10 min A,B.Pharmacological managementAfter 4 weeks of the NRMP, dogs who displayed a flexion/extension locomotor pattern but were DPP− started pharmacological management in combination with the training protocol, after the owners’ consent was obtained.This pharmacological treatment was based on a potassium channel blocker, 4-AP ,84,85,86Supportive careAll the dogs had neurogenic bladders; therefore, their bladders were manually expressed 3–4 times/day. Urine was monitored daily for odor and color changes, and in the case of suspected urinary tract infection (UTI), urine culture (by cystocentesis) and specific antibiotic treatment were performed. Only two dogs were UTI-positive and required antibiotic therapy with water consumption control. The dogs were maintained on a full-time hospitalization regimen and rested in soft beds. Feeding was performed three times/day (intake increase of 30%) and a hydric support of 100–120 mL/kg/day was provided after resistance training (training intended not to reach the maximum activity within an extended time period) that was alternated with fortification training (training intended to achieve the maximum activity within a short time period) according to the dog’s needs. All were trained during the day, starting at 9:00 a.m. and finishing at 7:00 p.m., assisted only by veterinarians and veterinary nurses that were CCRPs.During full-time NRMP, the dogs were evaluated neurologically every 3 to 7 days in order to make necessary changes concerning exercise duration and intensity. This evaluation included the assessment of the flexor reflex, flexion/extension locomotor pattern, postural standing, and the OFS , includiOutcomes were performed at 15, 30, 45, 60, 75, and 90 days, and they were followed up after 8–10 days, 1 month, and 6 months by the same CCRP instructor/examiner, based on the neurorehabilitation examination criteria and OFS.The primary outcome parameters of this study were the ambulatory status with an OFS ≥11, which indicates ambulation for DPP+ dogs. Regarding DPP−, ambulatory dogs will show spinal reflex locomotion (SRL). DPP− dogs without ambulatory status will show no spinal reflex locomotion (NSRL). Dogs with NSRL demonstrated the flexion/extension pattern but without the ability to rise.A secondary outcome was the pain perception assessment, evaluated on the tip and base of the tail and the perineum region (S1 and S2 dermatomes). Deep Pain Perception was assessed with 12 cm Halsted forceps in the lateral and medial digits of both pelvic limbs in a controlled environment regarding noise and the circulation of people.The evolution of the 16 chronic dogs was recorded after HWP and NRMP implementation, as illustrated in the flow diagram, as well as at follow-up. All the dogs were medically discharged when they achieved ambulation, with subsequent assessment by long-term follow-up after 8–10 days, 1 month, and 6 months .® . Categorical data were described as frequencies and proportions . The collected data included breed, sex, age, weight, lesion neuro-location, DPP presence, OFS at admission and at each time point , ambulatory status, time when ambulation was achieved, time of medical discharge, follow-ups , and the presence of concurrent events. Concurrent events during the NRMP included: pressure ulcers, alveolo-interstitial pulmonary disease, gastrointestinal disease, and bacteria/fungal dermatitis.Database and statistical analysis were performed using Microsoft Office Excel 2016The NRMP was applied to nine DPP− dogs (OFS 0) and seven DPP+ dogs (OFS 1) for a maximum period of 90 days. All the dogs had been paraplegic for more than 3 months after IVDD surgery and HWP. Dogs who achieved ambulation were discharged, as demonstrated in n = 16) paraplegic dogs, of which 56% (9/16) were DPP− and 44% (7/16) were DPP+.As mentioned above, data were collected regarding breed, age, gender, weight, neuro-location, and the presence of DPP. Moreover, this study included 100%  of dogs. No concurrent events were observed during the NRMP.Among the nine DPP− dogs that were admitted with an OFS of 0, 78% (7/9) were discharged with SRL and 22% (2/9) remained with NSRL.On day 15 and day 30, the dogs showed no significant evolution. Adverse events, such as vomiting, diarrhea, and seizures, were not seen in any of the dogs receiving 4-AP medication.At outcome day 45, two dogs (22% (2/9)) obtained SRL with medical discharge at day 60. On day 60, two more dogs (22% (2/9)) obtained SRL with medical discharge at day 90. Three more dogs (33% (3/9)) achieved SRL at outcome day 75, remaining until day 90. Only two dogs (22% (2/9)) remained with NSRL on day 90. The appearance of SLR occurred only after day 30, which may have been coincidental or may have been due to the contribution of 4-AP to their recovery.At the 8–10-day follow-up, all nine dogs maintained similar neurological status, as well as at the 1-month follow-up, although one dog missed their follow-up appointment. At the 6-month follow-up, only six dogs attended, and they all maintained the same neurological status. The evolution of the ambulatory status and medical discharge statistics are presented in The mean time in which ambulation was achieved, in DPP− dogs, was 62 days (median 60 days) and all regained automatic micturition.Seven DPP+ dogs were admitted with an OFS of 1, and 100% were discharged with ambulatory status.On outcome day 15, all dogs showed an improvement in the OFS (range 3 to 9) and two dogs achieved ambulatory status at outcome day 30: one with OFS 13 and the other with OFS 11. The first one was medically discharged on day 30, and the second only on day 60.n = 6), two more dogs achieved ambulatory status with an OFS of 11, and on outcome day 60 (n = 6), all the dogs were ambulatory, with two medical discharges with an OFS of 13.On outcome day 45 (n = 4), three dogs had OFS 12 and one had OFS 11. On day 90, the former improved to OFS 13 and the latter to OFS 12. The OFS results were registered during the outcomes, as shown in On outcome day 75 (The mean time to achieve ambulation in DPP+ dogs was 47 days (median 45 days) and all achieved voluntary micturition.The evolution of the ambulatory status and medical discharges are presented in At the 8–10-day follow-up, all seven dogs maintained similar OFS results, and at the 1-month follow-up, two dogs improved to an OFS of 14. At the 6-month follow-up, one dog did not attend, two improved to an OFS of 14, and one improved to an OFS of 13. The evolution of mean OFS results since medical discharge and during follow-up is represented in In this study’s population, the median age was 4 years old and 31% (5/16) of the dogs were French Bulldogs, which can be explained by the fact that this breed is fixed for the CFA12 FGF4 retrogene at a significantly younger age, as reported by Dickinson et al. (2020) , with a Regarding this study’s population, the mean weight was 7 kg, in line with most recent studies ,92,93. TIn the present study, these dogs had a chronic spinal injury that was present more than 3 months after surgery. Thus, the positive evolution in the locomotor pattern to achieve ambulation may indicate the benefits of the NRMP . This tyThe NRMP implemented was based on locomotor training, which included land treadmill by body weight supported treadmill training (BWSTT), UWTM, and kinesiotherapy exercises , as receThe land BWSTT enhances muscle endurance, promotes the rhythmic activity of the spinal network, and increases the stimulation of the remaining descending/ascending pathways ,100. LocIn this case series study, the results showed that in DPP– dogs, the neurological deficits improved after implementation of the pharmacological approach, with one dog achieving SRL 15 days after starting 4-AP, i.e., at the 45-day outcome. Moreover, 78% of ambulatory DPP− dogs achieved SRL by the 75-day outcome, i.e., within two and a half months. Thus, the mean time to achieve ambulation in DPP− dogs was 62 days and all achieved automatic micturition. At follow-up, all dogs maintained similar neurological status, due to the spinal cord’s memorization of the locomotor pattern. These results suggest that more studies are needed in this field.Furthermore, it is important to mention that, to the best of our knowledge, there is no study that unites the effects of intensive locomotor training, electrical stimulation, and 4-AP administration. Therefore, this is the first case series study that may prove the relevance of a multimodal approach.Lewis and colleagues (2019)  administIn order to be able to repeat this NRMP, it is necessary to use an electrical stimulation device, a land treadmill, and, ideally, a UWTM  ,105,106.In our dogs, pharmacological management was only applied within a period of two months. The possible lack of response may suggest that there is a limited functional effect of 4-AP in chronic SCI dogs when the neuronal matter is already in a state of hyperexcitability ,112. HowIn chronic SCI, the axons fail to regrow, and this axonal regrowth could be inhibited by scar tissue formation . The gliA recent analysis that assessed transcranial magnetic motor evoked potential suggested that spinal locomotion could be associated with remaining intact conduction through the descending motor tracts .Granger et al. (2012)  and Hu eHu et al. (2018)  concludeLocomotor training activity after SCI is a well-established modality to be applied after SCI, promoting the recovery of stepping in cats ,125. TheAll DPP− dogs in this study manifested some spinal network plasticity, allowing a flexion/extension locomotor pattern with a fast but modulated flexor reflex and crossed extensor reflex, characteristic of the NSRL ,127, whiIn the chronic phase, mainly in DPP− dogs, there is no standard treatment to regain ambulation, aiming to promote synapse plasticity, increase axonal sprouting, and lead to regeneration . The exiIt was shown that neurogenic induction, in both canines and humans, with dental pulp stem cells  could cause morphological changes .In rats with spinal cord transection, the combination of NSPCs with electroacupuncture has been shown to improve neurological evolution and regeneration ,137. SimIn Prado et al. (2019) , in 16 dCompared to the present study, the results described above may be related to the fact that locomotor training should be performed every day in order to induce plasticity in the reflex pathways ,139,140.One other study that used adipose tissue mesenchyme stem cells (AT-MSCs) demonstrated a regenerative function, either by mediating replaced lost cells, such as oligodendrocytes, which would facilitate the injured axons’ remyelination, or by mediating astrocytes. Therefore, some degree of neuroplasticity was evolved in axonal sprouting and the formation of new circuits. Moreover, they reported that the locomotor recovery was obtained within 8 weeks, with a tendency to stabilize between 8 to 16 weeks .In the present study, 100% of the DPP+ dogs achieved ambulatory status at clinical discharge. On day 15, all dogs had improved but only two achieved ambulation by day 30, the time point at which an OFS peak is achieved, as seen in Regarding DPP− dogs, there was no DPP recovery, in contrast to the acute dogs, who usually regain 41–62% of ambulation ,148,149.Since this was a case series study, we suggest that more studies are needed in this area, given the clinical interest and the need to be open to multidisciplinary approaches, mainly on post-surgical DPP− chronic dogs. This study’s population was difficult to obtain due to the strict inclusion criteria, because chronic post-surgical IVDD Hansen type I paraplegic dogs do not typically survive. We propose a multimodal approach in order to achieve ambulation, decrease the need to perform euthanasia, and promote the quality of life of these dogs.Thus, limitations included the small number of dogs, due to the criteria mentioned above, and the lack of control cases, which is a significant limitation of this study. The appearance of the SRL may have been due to spontaneous recovery, coincidental, or due to the contribution of 4-AP, and a control group would be required in order to assess this.Future controlled studies with a larger number of dogs with chronic DPP+ or DPP− incomplete recovery are needed. It would also be interesting to continue this study considering the association between the NRMP, stem cell administration, and/or a pharmacological management protocol with 4-AP.This prospective case series clinical study proposes a neurorehabilitation multimodal protocol (NRMP) as a safe and feasible therapeutic approach that was applied in all 16 chronic post-surgical SCI dogs without any side effects.n = 9) could not recover DPP within the 90 days of the NRMP, but 78% (7/9) were able to achieve ambulation by SRL, with a mean time of 62 days. Thus, it is suggested that this type of ambulation may be possible with a multidisciplinary protocol with the influence or not of spontaneous recovery. Moreover, it is possible that dogs that were admitted in a more severe state (DPP−) may simply take longer to respond to the non-pharmacological rehabilitation modalities when compared to DPP+ dogs.Chronic dogs with OFS 0/DPP− (n = 7) were able to achieve 100% of ambulation within a mean period of 47 days, suggesting the need for a longer period of time when compared to the average acute dogs who are submitted to similar rehabilitation approaches.On the other hand, dogs with OFS 1/DPP+  of ambulation, usually could not persevere. Thus, an NRMP may be an important therapeutic strategy to reduce the need for euthanasia in the clinical setting. This study should be continued, given all the limitations mentioned.