Main Research Findings

1) Treatment with Semax has been shown to reduce instances of behavioral and neurochemical alterations after neonatal exposure to SSRIs.

2) Semax administration was shown to activate the transcription of neurotrophins and receptor genes following periods of cerebral ischemia.

Selected Data

1) This study conducted by the research team of Glazova et al investigated the effects of the peptide Semax and the selective serotonin reuptake inhibitor (SSRI) fluvoxamine on early brain development, anxiety-related behavior, cognitive performance, and neurochemical profiles in Wistar rats. Wistar rat pups of both sexes, weighing 250–300 grams, were used. These rats were housed under controlled environmental conditions with a 12-hour light/dark cycle, food and water available ad libitum. Female and male rats were mated in a 3:2 ratio for three days, and their offspring were raised with their mothers until weaning on postnatal day 31. The day of birth was recorded as postnatal day 0, and a total of 19 litters were used. On postnatal day 1, the pups were weighed and randomly assigned to treatment groups, with at least one pup from each litter represented in every experimental condition. Daily weighing from postnatal day 1 to postnatal day 28 allowed precise dosing based on body weight [1].
Two compounds were studied: fluvoxamine maleate (FA), a commonly used SSRI, and Semax, a synthetic heptapeptide (MEHFPGP) with purported neuroprotective properties. FA was dissolved in sterile distilled water at a concentration of 5 mg/ml and administered intraperitoneally at 10 mg/kg from postnatal day 1 to postnatal day 14. This developmental window was chosen to model the late gestational stage in human brain development, a critical period during which SSRIs are known to exert lasting neurobehavioral effects. The dosage and route of FA administration were consistent with previous studies examining early-life SSRI exposure. Semax was administered intranasally at 0.05 mg/kg from postnatal day 15 to postnatal day 28, based on prior studies that demonstrated its efficacy and tolerability in neonatal and adult rats.
The research was divided into two main experimental series. In the first series, five litters were split into three groups: intact control (IC), control (CON), and Semax (SEM). The IC group received daily handling only, while the CON and SEM groups were injected with sterile water to act as a vehicle, from postnatal day 1 to postnatal day 14. From postnatal day 15 to postnatal day 28, the CON group received intranasal vehicle administration and the SEM group received intranasal Semax. This setup allowed researchers to examine the standalone effects of Semax, distinct from any pharmacological intervention [1].
In the second experimental series, 14 litters were divided into control (CON), fluvoxamine (FA), and fluvoxamine + Semax (FA-SEM) groups. The CON group received vehicle only during both treatment periods, while the FA group received FA from postnatal day 1 to postnatal 14 and vehicle from postnatal day 15 to postnatal day 28. The FA-SEM group received FA during the first period followed by Semax in the second period. This design modeled early-life SSRI exposure and allowed assessment of whether Semax could mitigate potential long-term neurobehavioral effects [1].
A range of behavioral tests were employed during the second month of life to evaluate anxiety, cognitive ability, and motivation. The elevated plus maze test was conducted on postnatal day 31 and postnatal day 60 to assess anxiety-related behavior. The maze consisted of two open and two closed arms elevated above the floor, and rats were allowed to explore for three minutes. Their activity was recorded and analyzed by observers blinded to the treatment groups. The test was performed under two different lighting conditions, dim light and bright, high-contrast lighting, to vary the level of aversiveness
The passive avoidance test, conducted from postnatal day 34 to postnatal day 37, evaluated memory retention. In this test, rats were trained to avoid entering a dark compartment where they previously received a mild foot shock. Retention was tested 72 hours later by measuring the latency to enter the dark compartment and total time spent in the light compartment [1].
Cognitive performance was also measured using a food-motivated maze task between postnatal day 42 and postnatal day 46. This maze required the rats to navigate a series of corridors to reach food reinforcement. After a 24-hour food deprivation, rats were allowed to explore the maze during a 30-minute adaptation session. Over the next four days, they completed five trials per day, during which latency to find food, the number of correct trials, and overall response time were recorded. To control for individual differences in food motivation, a separate food motivation test was conducted on postnatal day 49, in which rats were deprived of food for 20 hours and then observed for latency to eat, time spent eating, and total food consumed [1].
To evaluate potential neurochemical changes underlying the observed behaviors, brain tissues were collected on either postnatal day 32 or postnatal day 61. Rats were decapitated, and the frontal cortex, hypothalamus, hippocampus, and striatum were extracted, weighed, and frozen. These samples were later analyzed using high-performance liquid chromatography with electrochemical detection to quantify levels of dopamine, norepinephrine, and serotonin, as well as their major metabolites: 5-hydroxyindoleacetic acid (5-HIAA) and 3,4-dihydroxyphenylacetic acid (DOPAC). The tissue was homogenized in perchloric acid with an internal standard and centrifuged before analysis. Separation was achieved with a C18 reverse-phase column, and quantification was performed by comparing peak areas to known standards [1].
2) This study investigated the effects of the neuroprotective peptide Semax and its core fragment PGP (Pro-Gly-Pro) on gene expression in the rat brain following focal cerebral ischemia. The researchers aimed to understand how these treatments influence molecular responses during the acute and subacute stages of ischemic injury. The study employed a well-established model of permanent middle cerebral artery occlusion in adult male Wistar rats and analyzed changes in mRNA expression in the cortical tissue using both conventional and real-time reverse transcription polymerase chain reaction techniques [2].
Male Wistar rats, weighing between 270–320 grams, were housed with unrestricted access to food and water under natural light conditions. The animals were randomly assigned to nine treatment groups, each consisting of 15 to 17 rats. These included non-operated control groups treated with saline, Semax, or PGP; sham-operated control groups receiving the same treatments; and experimental groups that underwent focal cerebral ischemia followed by administration of saline, Semax, or PGP. Each group was further subdivided into time-point categories of 3, 24, and 72 hours post-treatment, with a minimum of five animals per subgroup.
Focal cerebral ischemia was induced by permanent occlusion of the distal portion of the left middle cerebral artery using electrical coagulation, performed under 300 mg/kg of chloral hydrate anesthesia. Sham-operated animals underwent the identical surgical procedures without actual arterial occlusion. Treatments including saline, 100 µg/kg Semax, or 37.5 µg/kg PGP, were administered intraperitoneally at 15 minutes and 1 hour after surgery. Animals in the 24-hour groups received additional injections at 4 and 8 hours, while those in the 72-hour groups received eight total injections spaced at 4–8 hour intervals up to 56 hours post-surgery. At the end of each designated time point ranging from 3, 24, or 72 hours, animals were euthanized under ethyl ether anesthesia, and the cortical tissue from both hemispheres was quickly excised and frozen in liquid nitrogen for molecular analysis [2].
In summary, this study employed an experimental design used to assess how Semax and its core peptide PGP influence gene expression in the brain following ischemic injury. By using standardized methods for inducing cerebral ischemia, isolating RNA, synthesizing cDNA, and performing qPCR, the researchers ensured high-quality, reproducible data. The inclusion of both sham-operated and non-operated control groups allowed for the distinction between effects due to surgery itself and those specifically caused by arterial occlusion. Multiple time points allowed the researchers to monitor gene expression changes during the early, middle, and late phases of the ischemic response. The findings from this study are expected to shed light on the molecular mechanisms underlying the neuroprotective actions of Semax and PGP, which may ultimately support their use in the treatment of ischemic stroke and other neurological conditions [2].

Discussion
1) The experimental study performed by researchers Glazova et al explored the behavioral and neurochemical effects of neonatal exposure to FA, an SSRI, and the potential therapeutic role of Semax, a synthetic peptide, in Wistar rats. The research was divided into two series of experiments that focused on various behavioral tasks and neurochemical analyses at different developmental stages. It assessed how early-life exposure to FA, with or without subsequent Semax treatment, affected anxiety-like behavior, learning and memory, motivation, and levels of key brain neurotransmitters [1].
The elevated plus maze test was used to assess anxiety-like behavior on postnatal days 31 and 60. The elevated plus maze test consists of open and closed arms elevated above the floor, with testing performed under bright and dim lighting to vary the stress level. Across all experiments, rats consistently spent more time in the open arms under dim light, indicating lower anxiety in less aversive conditions. Sex did not significantly affect elevated plus maze test performance, so results were presented for all animals regardless of sex.
On postnatal day 31, comparisons among the IC, CON, and SEM groups revealed no significant differences in elevated plus maze test performance under either lighting condition. This indicated that neither handling nor Semax administration alone affected anxiety-related behavior in adolescent rats. In contrast, rats exposed to FA showed increased anxiety under bright lighting conditions, evidenced by significantly reduced time spent in the open arms and fewer entries into the closed arms compared to controls. The FA-SEM group showed trends toward improvement, with increased open arm time compared to the FA group, though not statistically significant. Under dim lighting, no group differences were observed, suggesting that FA’s anxiety-inducing effects manifested mainly in high-stress environments and that Semax may partially mitigate these effects [1].
On postnatal day 60, similar behavioral patterns were observed. FA-treated rats again displayed heightened anxiety under bright lighting, with significantly reduced open arm time. The FA-SEM group showed a non-significant trend toward normalization of behavior, suggesting some lasting benefit of Semax. As with postnatal day 31, no behavioral differences were seen under dim light conditions, and sex did not influence outcomes. These findings imply that neonatal FA exposure increases anxiety in a context-dependent manner and that Semax may attenuate these long-term behavioral disruptions [1].
In the passive avoidance test, used to assess memory retention, there were no significant group differences in initial training latency or performance during the retention test, indicating that FA exposure did not impair basic associative learning. However, in the food-motivated maze task, which evaluates learning ability using a route-based design with food reinforcement, rats in the FA-SEM group performed significantly better than those in the FA group on the first training day. Specifically, they completed more correct trials and showed shorter latency and reaction times. This suggests that while FA impaired cognitive flexibility or goal-directed learning, subsequent Semax treatment improved performance.
In the food motivation test, rats were deprived of food and then given access to a food pellet. Males consumed more food than females, but there were no significant differences between treatment groups in latency to eat, total feeding time, or food consumed. This indicated that neither FA exposure nor Semax treatment affected overall food motivation, and observed differences in maze performance were not due to altered hunger levels [1].
To understand the neurochemical basis of these behavioral effects, the study measured levels of key monoamines including serotonin, norepinephrine, and dopamine, as well as their metabolites in multiple brain regions at postnatal day 32 and postnatal day 61. No sex differences were found in any biochemical measure, but inter-litter variability was significant and controlled by normalizing values to litter averages.
At postnatal day 32, neonatal FA exposure significantly elevated levels of serotonin and its metabolite 5-HIAA in the hippocampus and striatum compared to controls, indicating increased serotonergic activity. These effects were not present in the FA-SEM group, suggesting Semax normalized serotonin system activity in these regions. Similarly, in the hypothalamus, 5-HIAA levels were elevated in both FA and FA-SEM groups, indicating persistent serotonergic changes in this region, regardless of Semax treatment. Overall, these findings suggest that FA disrupts serotonergic signaling in key brain regions, and Semax may reverse or buffer many of these alterations [1].
At postnatal day 61, long-term changes in biogenic amines persisted in certain brain areas. In the frontal cortex, FA-treated rats exhibited significantly lower serotonin levels compared to controls, while serotonin levels in the FA-SEM group remained normal. In the hippocampus, there were trends toward increased norepinephrine and 5-HIAA levels in the FA group, both of which were reduced to near-control levels by Semax treatment. Norepinephrine levels in the hypothalamus were also elevated by FA but were not significantly different in the FA-SEM group, though Semax did not fully normalize hypothalamic norepinephrine. No significant group differences were found in the striatum at this age [1].
Taken together, the data suggest that early-life FA exposure causes region-specific and time-dependent disruptions in monoaminergic signaling, particularly affecting serotonin and norepinephrine systems. These alterations correspond with increased anxiety and impaired learning behaviors. Importantly, chronic postnatal Semax administration appears to mitigate many of the behavioral and neurochemical abnormalities induced by FA, indicating its potential as a therapeutic agent to counteract adverse effects of early SSRI exposure [1].
2) This study completed by Dmitrieva et al examined the expression patterns of neurotrophins and their receptors in the rat brain following focal cerebral ischemia, with and without treatment by Semax or its active fragment PGP. The objective was to determine how ischemia, along with neuropeptide treatment, affects the transcription of key neurotrophic factors and their receptors during the acute and subacute phases of brain injury [2].
Focal cerebral ischemia was induced by permanent middle cerebral artery occlusion, which produced localized damage in the ipsilateral frontoparietal cortex. Neurotrophin gene expression, including brain-derived neurotrophic factor (Bdnf), nerve growth factor (Ngf), and neurotrophin-3 (Nt-3), were evaluated in the affected cortical areas of ischemic rats, as well as in sham-operated and non-operated controls [2].
Three hours after surgery, ischemic rats showed a marked decrease in Bdnf mRNA levels in the cortex compared to both sham-operated and non-operated animals. Similarly, sham-operated rats also exhibited reduced Bdnf expression, indicating that surgical stress alone may suppress Bdnf transcription temporarily. However, Ngf mRNA levels were elevated in the ischemic cortex at this early time point, suggesting a rapid upregulation of this neurotrophin in response to injury. No significant changes were observed in Nt-3 mRNA expression at 3 hours in either ischemic or sham-operated animals.
At 24 hours post-occlusion, Bdnf expression significantly increased in the ischemic cortex, exceeding the levels seen in both control groups. This rebound suggests a delayed neurotrophic response potentially aimed at promoting repair. In contrast, Nt-3 mRNA levels declined in ischemic rats, while Ngf mRNA levels remained elevated. These changes reflect the dynamic, gene-specific regulatory patterns that occur in the post-ischemic brain. By 72 hours, changes in neurotrophin expression were more limited. Only Nt-3 showed a significant increase in ischemic rats compared to sham-operated controls. Bdnf mRNA levels in sham-operated rats increased by 72 hours but not significantly, suggesting partial recovery from surgical stress [2].
Semax treatment had distinct effects on neurotrophin expression. In non-operated rats, Semax did not alter Bdnf or Ngf mRNA levels at any time point, but it did reduce Nt-3 expression at 24 hours. However, in sham-operated animals, Semax increased Bdnf mRNA levels 24 hours post-surgery, indicating that it may help mitigate surgical stress. In ischemic rats, Semax elevated Bdnf mRNA expression as early as 3 hours after occlusion, potentially enhancing the neuroprotective response. Semax also modulated Nt-3 expression: it increased Nt-3 levels 24 hours post-occlusion but decreased them again at 72 hours. For Ngf, Semax consistently elevated its mRNA expression in the ischemic cortex at 24 and 72 hours, suggesting a sustained upregulation in response to injury [2].
PGP treatment produced a different pattern of effects. In non-operated rats, PGP reduced Bdnf expression at 3 hours but had no effect on Nt-3 or Ngf expression. In sham-operated rats, PGP increased Bdnf expression at 24 hours but reduced it again at 72 hours. It also increased Nt-3 expression at 72 hours and decreased Ngf expression at both 3 and 72 hours post-surgery. In ischemic rats, PGP increased Bdnf mRNA levels at 3 hours, paralleling the effects of Semax. At 24 hours, PGP led to a dramatic (over fourfold) increase in Ngf expression, indicating a strong neurotrophic response. However, PGP did not affect Nt-3 expression in ischemic rats at the selected time points.
The study also assessed the expression of neurotrophin receptors—TrkA, TrkB, and TrkC—in the cortex after ischemic injury. Three hours post-occlusion, TrkA mRNA levels decreased significantly in the ischemic cortex compared to sham controls. However, by 24 hours, TrkA expression had increased, surpassing both sham-operated and non-operated control levels. At 72 hours, sham-operated rats exhibited a significant decrease in TrkA expression. TrkB and TrkC expression levels were both reduced in ischemic animals 24 hours post-occlusion compared to controls, indicating a transient suppression of these receptors during early recovery [2].
Semax did not significantly alter Trk gene expression in rats without surgery. However, it reduced TrkA and TrkB mRNA levels in sham-operated animals at 3 hours. In ischemic rats, Semax treatment significantly increased TrkC and TrkA mRNA expression at 3 hours, which may reflect its early neuroprotective mechanisms by promoting receptor availability for neurotrophin signaling [2].

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

Citations

[1] Glazova NY, Manchenko DM, Volodina MA, et al. Semax, synthetic ACTH(4-10) analogue, attenuates behavioural and neurochemical alterations following early-life fluvoxamine exposure in white rats. Neuropeptides. 2021;86:102114. doi:10.1016/j.npep.2020.102114

[2] Dmitrieva VG, Povarova OV, Skvortsova VI, Limborska SA, Myasoedov NF, Dergunova LV. Semax and Pro-Gly-Pro activate the transcription of neurotrophins and their receptor genes after cerebral ischemia. Cell Mol Neurobiol. 2010;30(1):71-79. doi:10.1007/s10571-009-9432-0

Effects of Semax on Dopamine Expression

Semax is a heptapeptide that is considered to be an analog of the N-terminal fragment of adrenocorticotropin that has been shown to have various neuroprotective effects examined through several animal-based studies.

A study conducted by Levitskaya et. Al examined the effects Semax has on dopaminergic systems in the brain. White rats were given the neurotoxin MPTP in order to induce lesions on the dopaminergic centers. MPTP was given to the rats in 25 mg/kg doses and was shown to cause an increase in anxiety and a decrease in activity in the rats. Following the administration of the neurotoxin MPTP, the rats were given Semax at doses of 0.2 mg/kg. The study concluded that this dose of Semax greatly reduced the effects of the lesions caused by the neurotoxin. Overall, Semax acted in a protective manner against the lesions in the dopaminergic centers of the brain (https://link.springer.com/article/10.1023/B:NEAB.0000018752.59465.28).

Effects of Semax on Anxiety and Depression

As the positive benefits that Semax has on dopamine expression have already been discussed, further studies have built on that research to draw conclusions and perform studies on the effects Semax has on anxiety and depression.

In a study conducted by Leviskaya et. Al, rats were observed in a control group and in a group receiving treatment with cholecystokinin-tetrapeptide (CCK-4). Before administering the CCK-4, rats were administered Semax in doses of 50 and 500 microg/kg. 15 minutes after administration of Semax, the rats were given CCK-4 in doses of 400 microg/kg. Injection of CCK-4 caused increased levels of anxiety and depression in the rats which were confirmed through maze tests and forced swimming tests, respectively. In the rats treated with both CCK-4 and Semax it was found that administration of Semax reduced and normalized the neurodivergent behavior the rats were exhibiting, indicating that it could play a role in reducing levels of anxiety and depression when levels are elevated (https://link.springer.com/article/10.1134/S1062359010020147).

Effects of Semax on Experimental Ischemia

In addition to its effects on dopaminergic centers of the brain, Semax has also been shown to positively affect the morphology of cells within the nervous system that was damaged by ischemia in rats. A study conducted by Stavchansky et, Al showed that when treating the rats with Semax the capillary network was activated. This led to increased proliferation of blood vessel endothelium, neuroglia, and various different progenitor cells. Additionally, Semax was able to reduce the effects of ischemia in the brain and lessen the damage to the nervous tissue (https://link.springer.com/article/10.1007/s12031-010-9421-2).

A similar study conducted by Yakovleva et. Al found similar results as the previous experiment. The study examined the effects that Semax has on overall ischemia in the cerebral portion of the brain that was caused by occlusion of the carotid arteries. It was found that administering Semax to animals experiencing this type of ischemia led to an improvement in neurological functioning and survival of the neurons following the onset of ischemia (https://link.springer.com/article/10.1007/BF02433821).

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