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Abstract

Background Traumatic brain injury is a dangerous life threatening condition. This study examines the role of MLC901 in increasing neurogenesis. The aim of this study was to demonstrate the role of MLC901 in increasing neuron cell (neurogenesis) in rat with traumatic brain injury using the synaptophysin marker. Methods The synaptophysin levels were measured as a marker for neuron cell (neurogenesis) of brain nerve cells in Sprague-Dawley rats aged 10–12 weeks, weighing 200–300 g. All rats (n = 10) were performed as traumatic brain injury using The Modified Marmourou Model, then they were divided into 2 group, one group was given MLC901 (n = 5) and the other group was not given MLC901 (n = 5). The synaptophysin levels in both groups were assessed after 6 weeks and also carried out an examination of immnuhistochemical from the brain tissue of both groups. Results There was an increase in the number of neuron cells as evidenced by synaptophysin ihc staining in the rats given MLC 901 (Neuroaid II) compared to those without MLC 901. Synaptophysin levels were lower in the control group than in the MLC 901 group (81.6, SD: 13.52 vs 118.4, SD: 12.198, p = 0.062). Conclusion These research suggest that MLC901 can increase neurogenesis in traumatic brain injury and also appeared as synaptophysin antibody that binding to cytoplasm of neuronal cells in the rat brain.
Annals of Medicine and Surgery 60 (2020) 36–40
Available online 19 October 2020
2049-0801/© 2020 The Author(s). Published by Elsevier Ltd on behalf of IJS Publishing Group Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Experimental Research
Role of MLC901 in increasing neurogenesis in rats with traumatic
brain injury
Rohadi Muhammad Rosyidi
a
,
b
,
*
, Bambang Priyanto
a
,
b
, Andi Asadul Islam
a
,
Mochammad Hatta
a
, Agussalim Bukhari
a
, Krisna Tsaniadi Prihastomo
c
,
Rizha Anshori Nasution
d
, Rozikin
e
, Lale Maulin Prihatina
f
a
Medical Faculty of Hasanuddin University, Makassar, Indonesia
b
Department of Neurosurgery Medical Faculty of Mataram University, West Nusa Tenggara Providence General Hospital, Mataram, Indonesia
c
Department of Neurosurgery, Dr. Kariadi General Hospital Medical Center, Semarang, Center Java, Indonesia
d
Department of Neurosurgery, Pelamonia Hospital, Makassar, Indonesia
e
Research Unit, Faculty of Medicine, Al Azhar Islamic University, Mataram, Indonesia
f
Medical Faculty of Mataram University, Mataram, Indonesia
ARTICLE INFO
Keywords:
MLC 901
Neurogenesis
IHC Markers
Synaptophysin
Traumatic brain injury
ABSTRACT
Background: Traumatic brain injury is a dangerous life threatening condition. This study examines the role of
MLC901 in increasing neurogenesis. The aim of this study was to demonstrate the role of MLC901 in increasing
neuron cell (neurogenesis) in rat with traumatic brain injury using the synaptophysin marker.
Methods: The synaptophysin levels were measured as a marker for neuron cell (neurogenesis) of brain nerve cells
in Sprague-Dawley rats aged 1012 weeks, weighing 200300 g. All rats (n =10) were performed as traumatic
brain injury using The Modied Marmourou Model, then they were divided into 2 group, one group was given
MLC901 (n =5) and the other group was not given MLC901 (n =5). The synaptophysin levels in both groups
were assessed after 6 weeks and also carried out an examination of immnuhistochemical from the brain tissue of
both groups.
Results: There was an increase in the number of neuron cells as evidenced by synaptophysin ihc staining in the
rats given MLC 901 (Neuroaid II) compared to those without MLC 901. Synaptophysin levels were lower in the
control group than in the MLC 901 group (81.6, SD: 13.52 vs 118.4, SD: 12.198, p =0.062).
Conclusion: These research suggest that MLC901 can increase neurogenesis in traumatic brain injury and also
appeared as synaptophysin antibody that binding to cytoplasm of neuronal cells in the rat brain.
1. Introduction
Traumatic brain injury is a major cause of death and disability in
modern society, today this condition most often faced by neurosurgeons
related to advances in science and technology especially in industry and
transportation in large cities that were not accompanied by good road
construction [13]. In the past two years, researcher has experience the
use of neuroid drugs in patients with traumatic brain injury and it has a
signicant results. Many pathological processes contributes to traumatic
brain injury are targeted by neuroaids [4,5].
MLC 901 (Neuroaid II) is a traditional chinese medicine that facili-
tate the restoration of neuron circuits by its antioxidant properties,
initiating cell proliferation, and stimulation of axonal and dendritic
neuron circuits after traumatic brain injury. In rat models, neuroaids
have been shown to prevent cell death and stimulate new neurogenesis.
Rats that given neuroaids after ischemic injury showed increased in
survival, improved neurological recovery, improved cognitive function
and decreased neurodegeneration [5,6].
Neurogenesis or cell proliferation is the initial process of neuron
formation, then the neuron migrates and survives until it becomes
mature and integrates as a new neuron [7]. The neurogenesis process
starts from cell proliferation to migration and differentiates into
neuronal cells in the hippocampus, it is estimated to take approximately
4 weeks [8].
* Corresponding author. Departement of Neurosurgery Medical Faculty of Mataram University, West Nusa Tenggara Provience General Hospital, Mataram
Indonesia.
E-mail address: rha.ns2010@gmail.com (R.M. Rosyidi).
Contents lists available at ScienceDirect
Annals of Medicine and Surgery
journal homepage: www.elsevier.com/locate/amsu
https://doi.org/10.1016/j.amsu.2020.10.013
Received 14 September 2020; Received in revised form 7 October 2020; Accepted 8 October 2020
Annals of Medicine and Surgery 60 (2020) 36–40
37
Synaptophysin is an integral membrane protein located in the syn-
aptic vesicles and part of the neuroendocrine secretory granule mem-
brane that is recognized by monoclonal antibodies. Synaptophysin is a
broad-spectrum neuroendocrine marker, formed when vesicles fuse
with the presynaptic membrane. It is a specialized and sensitive marker
at the synaptic terminal of nerve cells [9,10]. This research is hypoth-
esized that the administration of neuroaid can increase neuronal cell or
neurogenesis in traumatic brain injury.
2. Methods and materials
This study examined the administration of MLC 901 to number of
neuronal cell (neurogenesis) after traumatic brain injury in serum and
brain tissue, then compared the responses between two groups; groups
that were given neuroaid and groups that were not given MLC 901.
2.1. Animals
This study was approved by the ethics commission of The Faculty of
Medicine, Hasanuddin University, with license number: 771/UN
4.6.4.5.31/PP36/2020. The surgical procedure was carried out asepti-
cally. Ten Sprague-Dawley rats, (aged 1012 weeks, weight 280300 g)
obtain standard food (Comfeed AD-2) and water until research occurs.
Rats were divided into two groups: (1) brain injury with MLC 901
administration, (2) brain injury without MLC 901 administration.
2.2. Treatment of brain injury and tissue retrieval
Ten Sprague Dawley rats with brain injury were randomly divided
into two groups: (1) with MLC 901 administration, and (2) without MLC
901 administration (placebo). Anesthetics are performed with diluted
ketamine (dose 310 mg/kgBW). The brain surgery is done by a corona
incision along center line of the head and then making a burr hole in
which a hole is drilled or scraped into the rats skull, until the dura mater
is exposed. The trauma was performed as The Modied Marmarou
Model [11,12], using 20 g of load then dropped from 20 cm of height,
through a tube [13,14]. The part of dura mater that has been exposed is
placed below the tube, so that the load falls on precisely at dura mater
and it is conrmed that the trauma has caused brain damage. One rat
was subjected to pathology examination (the result was bleeding from
the brain tissue), while the other rats were treated according to the
standard craniotomy procedure. The wound was sutured and antibiotic
ointment was applied. All surgical procedures are carried out aseptically
with the principle of sterility. After the procedure, all rats were treated
at room temperature in standard cages for recovery.
At 6 weeks after treatment, the rats were euthanized using 400 mg of
phenobarbital via parenteral (injection) [15,16], then the brain tissue
extraction performed by carniectomy. The brain tissue was immediately
frozen at 80 C until further processed through immuno-histochemical
examination using synaptophysin markers.
2.3. Administration of MLC 901
At 30 min after brain injury, MLC 901 was administered per sonde at
a dose of 68.4 mg/day until termination at sixth week.
2.4. Sampling and testing
Brain tissue samples were taken 6 weeks after MLC 901 adminis-
tration. The brain tissue was using parafn and hematoxylin eosin (HE)
staining method and was tested immunohistochemically using syn-
aptophysin markers.
2.5. Statistical analysis
Data are presented as mean ±SD. All data were processed and
analyzed using Excel 2013 and SPSS version 23 (IBM Corp. Released
2015. IBM SPSS Statistics for Windows, Version 23.0. Armonk, NY: IBM
Corp.). Number of Neuron Cell, with Synaptophysin markers staining
levels were analyzed by using the T Independent Test. P value less than
0.05 was considered statistically signicant.
3. Result
3.1. Subject characteristics
This study examines the role of MLC 901 in increasing neurogenesis.
By using a brain injury model in rats (Sprague Dawley), this study aims
to explain the benets of MLC 901 in increasing number of neuronal cell
(neurogenesis) after traumatic brain injury. Characteristics of subjects,
such as body weight, are listed in Table 1.
3.2. Synaptophysin levels
In the table above, it was found that synaptophysin levels were
higher in MLC901 administration (118.4) than without MLC 901
administration (81.6), and also there was a signicant difference (p =
0.002). These ndings can also be illustrated clearly in the boxplot chart
below.
3.3. Histopathological image
The histopathological image of rat brain tissue on immunohisto-
chemical examination with synaptophysin antibody stains can be seen in
Fig. 2 and Fig. 3 below.
4. Discussion
The traumatic brain injury is an injury to brain tissue, not due to a
degenerative or congenital process but because of an external impact
that can result in a decrease or change in the state of consciousness.
Brain injury is the leading cause of death from accidents under the age of
40. Each year around 10 million people worldwide are hospitalized for
brain injuries [17,18].
Traumatic brain injury is a complex condition involving primary and
secondary brain injury. Combined therapy could be a better treatment
strategy, using formulations that contain more than one active ingre-
dient. Traditional Chinese Medicine (TCM) has been recommended for
centuries to treat a wide variety of medical conditions. Traditional
Chinese Medicine which consists of several extracts of herbal medicine
has received attention from the medical world. MLC 901 (Neuroiad II)
have emerged as a promising treatment to support neurological recov-
ery. Several clinical trials and reports have established its safety prole
[19]. Research by Tsai et al., 2014, found that MLC 601 (Neuroaid I) had
a positive effect on reducing brain contusion in rat with traumatic brain
injury. Contusions due to traumatic brain injury are associated with
neurological motor decits, brain apoptosis, and activated microglia
[20].
The experimental animals used were Rattus Norvegicus strain,
Sprague Dawley rats, with an average body weight of 290.07 (±10.48)
grams; there is no signicant difference in the body weight of the sub-
jects. Any differences in the expression of dependent variable are
Table 1
Weight data of sprague dawley rats.
Rat Weight (g) P-value
Mean 290.7 0.155
SD ±10.48
The Levenes test of homogeneity in Sprague Dawley rat with brain injury ob-
tained p value >0.05, and it can be concluded that the weight of each rat is not
signicantly different.
R.M. Rosyidi et al.
Annals of Medicine and Surgery 60 (2020) 36–40
38
expected to be the result of neuroaid treatment in traumatic brain injury.
The aim of this study was to determine the effect of MLC 901 (neu-
roaid II) in increasing number neuronal cell (neurogenesis) in rat brain
tissue that had traumatic brain injury using immuno-histochemical ex-
amination with neuronal cell markers (synaptophysin markers). Syn-
aptophysin is a component of presynaptic vesicle membrane and as a
Fig. 1. Synaptophysin levels in rats with MLC 901 and without MLC 901.
Fig. 2. Normal immunohistochemical features of Sprague Dawley rat brain tissue. This gure shows pieces of brain parenchyma tissue, that consist of neuron cells
and granule cells. 100×magnication.
Fig. 3. Image of synaptophysin antibodies in rat
brain tissue. A). Without MLC901. Synaptophysin
antibody is not smeared on the cytoplasm of neuron
cell (arrow sign). There is no antibody binding be-
tween synaptophsyin and cytoplasm of neuron cell, so
that the cytoplasm remains blue. 400×magnication.
B). MLC 901 (Neuroaid II) administration. Immuno-
histochemical stain with synaptophysin antibodies.
Synaptophysin antibody smeared on the cytoplasm of
neuron cell (arrow sign). There is an antibody bond
between synaptophsyin and cytoplasm of neuron cell,
so that the color of the cytoplasm turns brown. 400×
magnication. (For interpretation of the references to
color in this gure legend, the reader is referred to the
Web version of this article.)
R.M. Rosyidi et al.
Annals of Medicine and Surgery 60 (2020) 36–40
39
marker of nerve cell in surgical neuropathology and most commonly
used neuronal cell markers. Synaptophysin is a sensitive marker of
neuron differentiation and neuroendocrine which can also be found in
nerve cell tumors such as medulloblastoma and Primitive Neuro-
Ectodermal Tumors (PNET) [21,22]. Synaptophysin play an important
role in maintaining the fusion of vesicles on the presynaptic neuronal
activity by provide the complement of the synaptobrevin-II molecule in
vesicles presynaptic. The absence of synaptophysin results in a pro-
gressive reduction of exocytosis presynaptic vesicle component [23,24].
In vivo research by Tarsa (2009), conclude that the synthesis of syn-
aptophysin secretion is regulated by neuron growth factors which then
act as a development regulator for trigeminal ganglion cells. Neuron
growth factor and synaptophysin together facilitate the transportation
of synaptic vesicles from the cell body to the presynaptic terminal for the
development of trigeminal ganglion cells [25].
MLC 901 derived from traditional chinese medicine, MLC 901 shown
to have neuroprotective and neurorestorative properties in preclinical
models of stroke, global cerebral ischemia and traumatic brain injury.
Neuroaid contains 9 herbal components namely Radix astragali, Radix
salvia miltiorrhizae, Radix paeoniae rubra, Rhizoma chuanxiong, Radix
angelicae sinesis, Carthamus tinctorius, Prunus persica, Radix poly-
galae, and Rhizoma acori tatarinowii [6].
MLC 901 have neuroprotective and neurorestorative effects that lead
to improved recovery of cognitive function in rats with traumatic brain
injury. This is a great base to explore the effect of neuroid therapy to
improve recovery of patients with traumatic brain injury [26].
In Table 2, there was an increase in the expression of synaptophysin
levels in MLC 901 group. These results indicate that the administration
of MLC 901 in trauamtic brain injury gives a good response to increasing
neurogenesis process. Previous Study from Quintard, H. et al., 2014
showed positive MLC901 effects were associated with an upregulation of
vascular endothelial growth factor (VEGF) as well as an increase of
endogenous hippocampal neurogenesis and gliogenesis around the
lesion. MLC901 reduced cognitive decits induced by TBI. Rats sub-
jected to TBI displayed a suppression of temporal order memory, which
was restored by MLC901. This work provides evidence that MLC901 has
neuroprotective and neurorestorative actions, which lead to an
improvement in the recovery of cognitive functions in a model of trau-
matic brain injury. Previous Clinical studies conducted on 32 patients
with moderate brain injury, showed the result of study were signicant
clinical outcome in MLC 601 (Neuroaids I) group compared to control
group and no side effects were found [26,27].
Immunistochemical examination of rat brain tissue is shown in
Fig. 3. There is a bond of synaptophysin antibody in the cytoplasm of
nerve cell in the group that given MLC 901 (neuroaid II) compared with
the group that was not given MLC 901. These results indicate that the
effect of MLC 901 can increase the neurogenesis process of nerve cells.
MLC 901 treatment stimulates gliogenesis and neurogenesis, which can
be helpful in inducing dynamic brain remodeling and leading a better
neurological recovery in the rst weeks after traumatic brain injury. The
positive effect of MLC 901 on neuronal plasticity (as characterized by
increased neurogenesis, neurite growth, axonal growth, dendritic
arborization and/or synaptogenesis) has been observed in focal and
global ischemia and correlates with functional recovery [5,26]. The
limitation of this study is that the research was carried out on experi-
mental animals so that it needs to be continued in the future with clinical
research involving neurogenesis biomarkers.
5. Conclusion
From the results and descriptions above, it can be concluded that the
administration of MLC 901 (neuroaid II) can increase number of
neuronal cell (neurogenesis) in a rat model with brain injury.
Ethical approval
All procedure for Animal experiment has been approved by Ethics
Commission Faculty of Medicine, Hasanuddin University, Number: 137/
UN4.6.4.5.31/PP36/2020.
Sources of funding
No funding or sponsorship.
Author contribution
RHA, BAM, AAI, MH and ASB wrote the manuscript and participated
in the study design. RHA, BAM, AAI, MH, NAA, ASB, RAN and RZ
drafted and revised the manuscript. RHA, AAI, BAM, MH, ASB and RAN
performed head trauma treatment and surgery. RHA, NAA, and RZ
performed bioinformatics analyses and revised the manuscript. All au-
thors read and approved the nal manuscript.
Registration of research studies
1. Name of the registry:
2. Unique Identifying number or registration ID:
3. Hyperlink to your specic registration (must be publicly accessible
and will be checked): None
Guarantor
Rohadi Muhammad Rosyidi.
Declaration of competing interest
The authors declare that they have no conict of interests.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.amsu.2020.10.013.
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R.M. Rosyidi et al.
... MLC 901 has neuroprotective and neuroproliferative effects and has been extensively described during in vitro and in vivo experiments using animal models. The remarkable effect of MLC 901 lies in its neurogenesis and neurorestoration effects rather than its neuroprotective effects [25][26][27]. ...
... All surgical procedures were performed aseptically by adhering to the principle of sterility. After the surgery and trauma, all animals were kept at room temperature for recovery and returned to their cages according to their groups [20,27,28,30]. ...
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... Further damage will cause the release of neurotoxic mediator directly or nitric oxide (NO) and cytokines indirectly. The release of vasoconstrictors (prostaglandin and leukotriene) and damage of microvascular structures caused by the adhesion of leucocyte and thrombocyte, blood-brain-barrier damage, and tissue edema will cause a decrease in tissue perfusion, thus increasing the severity of secondary brain injury [3][4][5]. Neuroinflammation in head injury is like a symphony and balance between proinflammatory and anti-inflammation responses [6,7]. This study evaluated homocysteine, TNFα, IL-10, and HMGB1 as a biological markers of inflammation in traumatic brain injury. ...
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Background Head injury or traumatic brain injury is the leading cause of mortality and morbidity. Many modalities of neuroprotection had been developed in brain injury but there was no much information regarding folinic acid's effect on neuroinflammation associated with homocysteine, TNFα, IL-10, and HMGB1. Objective This study aimed to investigate whether folinic acid has improving effect on head injury model. Method This study was done in the rat's head injury model using modified Marmarou weight drop model. Fifteen rats were randomized and grouped into 3 groups: Group A: Folinic acid (+), head injury (−); Group B: Folinic acid (−), head injury (+); Group C: Folinic acid (+), head injury (+). Folinic acid was administered intraperitoneally with a dose of 60 mg/m². Blood samples were taken immediately after head injury (H0), 12 h (H12), and 24 h (H24) after head injury from the lateral vein of tail. Serum level of homocysteine, TNFα, and IL-10 were measured using ELISA, and HMGB1 gene expression was measured with Real-Time RT-PCR. Results This study found serum level of homocysteine, TNFα, IL-10 and HMGB1 gene expression were markedly increased at all time points after head injury. Significantly lower level of serum homocysteine, TNFα, IL-10 and HMGB1 gene expression were found after 24 h treatment with folinic acid in group C compared to those in group B. Conclusion Folinic acid may have anti-inflammatory properties in traumatic brain injury by inhibition of serum level of homocysteine, TNFα, IL-10 and HMGB1 gene expression.
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Background A head injury is a very dangerous condition that threatens human life. This study examines the use of caffeic acid phenethyl ester (CAPE) in reducing cerebral edema in cases of head injury. The purpose of this study is to demonstrate whether CAPE can improve various parameters related to the expression of Aquaporin-4 (AQP4) mRNA and the serum AQP4 levels in rat subjects. Methods This is a randomized controlled study using a posttest-only control group design that uses experimental animals—specifically, male Rattus norvegicus (Sprague Dawley strain) rats aged 10–12 weeks and weighing 200–300 g. This study used a head injury model according to Marmarou (1994) with minor modifications to the animal model fixation tool. The parameters of the AQP4 mRNA were examined with real-time PCR, while serum AQP4 levels were examined with sandwich ELISA. Results The AQP4 mRNA expression in rats that were given CAPE was lower than those not given CAPE, both on the fourth and seventh days; serum AQP4 levels in rats that were given CAPE were also lower than those not given CAPE, both on the fourth and seventh days. Conclusion Administration of CAPE in a rat model with head injury can reduce cerebral edema, mediated by AQP4.
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Introduction Head injury is an injury or wound of the brain tissue due to external forces; it can cause a decrease or change in the status of consciousness. Many head injury models have used mice as experimental animals; the Marmarou model is the most famous and the most widely-used diffuse brain injury model. In this study, we slightly modified the Marmarou model. The purpose of this study is to help researchers examining head injuries in mice, especially those in developing countries who have limited facilities and infrastructure. Methods This experimental research uses animals models (Rattus novergicus, strain Sprague Dawley) that fit several criteria, including male, aged 10–12 weeks, and body weight of 200–300 g. This study involves a slight modification on the tube used, with a 20 cm-long weight of 20 g. The blood samples for the following assays of ELISA and brain tissue samples were collected at 24 h and 4, 5, 6, and 7 days post-trauma. Results A significant effect on the brain was seen with the Marmarou model modification, at a mass weight of 20 g and height of 20 cm, with 0.04 J energy produced. Changes were also seen in the histological features of brain tissue and the serum levels of AQP-4, F2 IsoPs, MPO, and VEGF from 24 h until 7 days after trauma. Conclusion This report describes the development of an experimental head injury approach modifying the Marmarou model that is able to produce a diffuse brain injury model in mice.
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Background: MLC601 is a natural product formulation from Chinese medicine that is extensively studied in ischemic stroke. Traumatic brain injury (TBI) shares pathophysiological mechanisms with ischemic stroke, yet there are few studies on the use of MLC601 in treating TBI. This Indonesian pilot study aimed to investigate clinical outcomes of MLC601 for TBI. Methods: This randomized controlled trial included subjects with nonsurgical moderate TBI allocated into two groups: with and without MLC601 over three months in addition to standard TBI treatment. Clinical outcomes were measured by the Glasgow Outcome Scale (GOS) and Barthel Index (BI) observed upon discharge and at months (M) 3 and 6. Results: Thirty-two subjects were included. The MLC601 group (n = 16) had higher GOS than the control group (n = 16) at all observation timepoints, though these differences were not statistically significant (p = 0.151). The BI values indicated a significant improvement for the MLC601 group compared to the control group at M3 (47.5 vs. 35.0; p = 0.014) and at M6 (67.5 vs. 57.5; p = 0.055). No adverse effects were associated with MLC601 treatment. Conclusion: In this cohort of nonsurgical moderate TBI subjects, MLC601 showed potential for a positive effect on clinical outcome with no adverse effects.
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Background Treatments to facilitate recovery after traumatic brain injury (TBI) are urgently needed. To examine the safety and potential effects of herbal supplement MLC901 (NeuroAiD IITM) on cognitive functioning following TBI, we conducted a nine‐month pilot, randomised placebo‐controlled clinical trial. Methods Adults aged 18‐65 years, 1‐12‐months post‐mild or moderate TBI, were randomised to receive MLC901 (0.8g capsules/day) or placebo for 6‐months. The primary outcome was cognitive functioning assessed by the CNS‐Vital Signs online neuropsychological test. Secondary outcomes included Cognitive Failures Rivermead Post‐Concussion Symptoms (neurobehavioral sequelae), Quality of Life after Brain Injury, Hospital Anxiety and Depression Scale, Fatigue Impact Scale and Glasgow Outcome Scale‐Extended (physical disability). Assessments were completed at baseline and 3, 6, 9‐month follow‐up. Linear mixed effects models were conducted, with the primary outcome time‐point of 6‐months. Results Seventy‐eight participants (mean age 37.5±14.8, 39 (50%) female) were included in the analysis. Baseline variables were comparable between groups (treatment group n=36, control group n=42). Linear mixed effects models controlling for time, group allocation, repeated measurements, adherence and baseline assessment scores, revealed significant improvements in complex attention (p=0.04, d=0.6) and executive functioning (p=0.04, d=0.4) at 6‐months in the MLC901 group compared to controls. There were no significant differences between the groups for neurobehavioral sequelae, mood, fatigue, physical disability or overall quality of life at 6‐months. No serious adverse events were reported. Conclusions MLC901 is safe and well tolerated post‐TBI. This study provides Class I/II evidence that for patients with mild to moderate traumatic brain injury, 6‐months of MLC901 improves cognitive functioning. This article is protected by copyright. All rights reserved.