The etiology of ASD is not well understood, though it likely involves genetic, immunologic, and environmental factors [9]. The dramatic increase in reported prevalence has encouraged an intense effort to identify early biological markers [10]. Such markers could allow for earlier identification and therapeutic intervention, contributing to improved prognosis [11]. In the current study, we speculated an important connection between SHH, BDNF pathways and oxidative stress. In our study, we demonstrated statistically significant increase in free radicals production (superoxide anion (O2) hydrogen peroxide (H2O2), and hydroxyl radicals (OH)), from whole blood and isolated human PMNLs in autistic children when compared to age and sex matched control. This increase was not related to the degree of autism, or to the age of affected child. In addition, we demonstrated higher serum level of SHH concentration, which was positively correlated with the degree of autism. Furthermore, we found a statistically significant higher level of BDNF in mild but not in sever autism.

Oxidative stress is a process caused by exposure to reactive oxygen intermediates, such as superoxide anion (O 2 −) hydrogen peroxide (H 2 O 2 ), hydroxyl radicals (OH) and nitric oxide (NO) which can damage proteins, nucleic acids and cell membranes. The ROS within the cells are neutralized by antioxidant defense mechanisms. SOD, catalase and glutathione peroxidase (GPx) are the primary enzymes involved in direct elimination of ROS, whereas glutathione reductase and glucose-6-phosphate dehydrogenase are secondary antioxidant enzymes, which help in maintaining a steady concentration of glutathione and NADPH necessary for optimal functioning of the primary antioxidant enzymes [12–15]. Under normal conditions, a dynamic equilibrium exists between the production of reactive oxygen species (ROS) and the antioxidant capacity of the cell [16, 17]. In pathological conditions, over production of OFR or less effective antioxidant enzymatic system, takes place, resulting in OFR overflow which leads to tissue damage. Oxidative stress is an important mechanism involved in brain damages, as consequences of exposure to reactive oxygen species (ROS) [15, 17].

Brain-derived neurotrophic factor (BDNF) is a small protein found throughout the central nervous system (CNS) and peripheral blood. BDNF is the most widely distributed neurotrophin in the CNS. BDNF plays a critical role in axonal and dendritic growth and guidance. In addition, BDNF participates in neurotransmitter release [18]. BDNF is involved in the survival and differentiation of dopaminergic neurons in the developing brain [18, 19] and plays an important role in the formation and plasticity of synaptic connections [20].

Within the nervous system, SHH protein is associated with development and patterning of the central nervous system [5, 21, 22]. Latest reports have signified a critical role played by SHH pathway in many neurological diseases. However, its exact role and the underlying mechanisms are still unclear. It has been reported that SHH expression is up-regulated prior to the induction of BDNF mRNA, and blocking SHH signals suppresses BDNF expression [23]. Considering the protective role of BDNF against oxidative stress [24], therefore, activation of the SHH pathway induces the increase of BDNF and results in neuroprotective to oxidative stress. BDNF belongs to the neurotrophin family that may affect neuronal survival and differentiation. SHH is a morphogen important for the embryonic development. Possible correlation between BDNF and SHH is less well studied. Hashimoto and his colleagues in 2008, demonstrated up-regulation of SHH expression, prior to the induction of BDNF mRNA in Schwann cells adjacent to the injured site in an animal model of sciatic nerve injury [23]. The same research group demonstrated causative relationship between the induction of SHH and BDNF, continuous administration of hedgehog inhibitor CPM to the injured site suppressed the increase of BDNF expression and, notably, deteriorated the survival of motor neurons in lumbar spinal cord [23].

Wu et. al. [25], demonstrated BDNF-induced up-regulation of SHH at both mRNA and protein levels, suggestive of involvement of a transcriptional mechanism. Furthermore, the protective effect of BDNF was abolished by SHH signaling inhibitor CPM. In addition, exogenous SHH-N alone mimicked BDNF action that was sufficient to attenuate 3-NP toxicity towards cortical neurons in a dose- and time-dependent fashion [25]. Lately, a protective action exerted by SHH has been revealed in several animal models of ischemia/reperfusion [26]. In addition, pretreatment of SHH has been shown to protect cardiomyocytes against hydrogen peroxide-induced cytotoxicity in vitro [27]. As a result, SHH may offer both anti oxidative and anti-apoptotic actions under appropriate circumstances. Metabolic stress induced by compromised mitochondria has been implicated in both acute and chronic neurodegenerative disorders such as ischemic stroke, Alzheimer’s disease, Parkinson’s disease (PD), and HD. It has also been shown that SHH reduces behavioral deficits induced by intrastriatal 6 hydroxydopamine (6-OHDA) lesion and suggests that SHH may be useful in the treatment of disorders that affect the nigrostriatal system, such as PD [28].

Systemic administration of several neurotrophins such as nerve growth factors and brain derived growth factor can attenuate neuronal damage induced by chemical hypoxia in vivo by a mechanism which may involve attenuation of oxidative stress in neonatal rat model [29].

Normal SHH pathway was found to be necessary for wound healing, and impaired coetaneous SHH signaling pathway contributes to impaired NO function and wound healing in diabetes. Delivery of exogenous SHH protein or its receptor agonists may provide an effective means in accelerating diabetic wound healing. Strategies aimed at augmenting endogenous SHH pathway may provide an effective means in ameliorating delayed diabetic wound healing [30].

An increase in SHH protein was initially reported in the gray matter from multiple sclerosis brains lesions [31] or in animal models of this pathology including Experimental Autoimmune Encepalomyelitis and cuprizone-induced demyelination [32, 33]. The clinical improvement and reduced demyelination driven by either interferon-b or triiodothyronine in several rodent models of demyelinating diseases are likely linked to an enhanced expression of SHH [32, 33]. It points out towards a possible protective effect of exerted by SHH and it is with agreement to our findings in autism. The most likely explanation for higher level of SHH in autistic examined in this study is as a result of increased oxygen free radicals production as a protective mechanism secondary to increase oxidative stress inside the autistic. The roles of the SHH signaling pathway in the CNS are gaining some interest lately, due to a multifunctional properties of SHH, ranging from the regulation of new cells production to the modulation of neuronal electrophysiological activity. The higher levels of OFR and consequently SHH, demonstrated in the current study, in mild ASD, resulted in an increase BDNF production as a protective mechanism. In severe ASD the further increase in OFR and SHH, produced a negative feed back response on the production of BDNF, as demonstrated by lower level of BDNF in severe but not in mild ASD. Existing data provide support for considering SHH signaling as an important mechanism in tissue-repair process in brain diseases, and as a target for novel therapeutic approaches for the treatment of brain disorders and in particular ASD.