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A novel pharmacological treatment concept for neuroprotection in severe traumatic brain injury—Two case reports

Abstract Severe traumatic brain injury (sTBI) is a major cause of death and disability worldwide, resulting in a significant individual and socioeconomic burden. Current treatment guidelines do not include any recommendations for neuroprotective or neuoregenerative drugs. Here, we present a combined treatment with Cerebrolysin and Citicoline in two cases. Both drugs are experimentally better than clinically proven in their own effectiveness, but there is almost no clinical data on the combination of the two. Our case study hints at a promising approach that may improve neurological outcome after sTBI. The first patient was a 29 years male motorcyclist suffered polytrauma in a high‐speed accident. He had severe bilateral chest trauma and fractures in both thighs and an sTBI. In addition to surgical and standard neurocritical care according to the evidence‐based guidelines, he was given neuroprotective therapy with Cerebrolysin (50 ml/day) and Citicoline (3 g/day), by continuous intravenous infusion (IV), for 21 days. The second patient was a 30 years male ski mountaineer who had suffered a fall over 300 m in open terrain. In addition to the sTBI, he had fractures in the cervical spine, ribs, pelvis, and lower extremities, as well as lung contusions and massive soft tissue trauma. After initial treatment in a local hospital, he was transferred to our department and received the same neuroprotective drugs, like all of our patients with sTBI. Considering the severity of the injuries (Injury Severity Score [ISS]: 43/50, Revised Trauma Score [RTS: 5.0304, 2.7794]) and the unfavorable outcome probability (Hukkelhoven Score) of 93.1% and 82.6%, the outcomes of both patients are surprisingly encouraging 1 year after the accident. They achieved a Glasgow Outcome Score of 6 and 5 and grades 2 and 4 on the modified Rankin Scale, respectively. Currently, both are able to take care of themselves in activities of daily life to a large extent. Neuroprotective drugs may improve the regeneration of cell membranes, improve blood brain barrier integrity, and reduce neuroinflammation leading to secondary damage to the injured brain. Our clinical experience and data suggest that the combined administration of Citicoline and Cerebrolysin may contribute to better recovery, without relevant side effects. However, it would be important to validate these results by means of a controlled, prospective study.

than clinically proven in their own effectiveness, but there is almost no clinical data on the combination of the two. Our case study hints at a promising approach that may improve neurological outcome after sTBI. The first patient was a 29 years male motorcyclist suffered polytrauma in a high-speed accident. He had severe bilateral chest trauma and fractures in both thighs and an sTBI. In addition to surgical and standard neurocritical care according to the evidence-based guidelines, he was given neuroprotective therapy with Cerebrolysin (50 ml/day) and Citicoline (3 g/day), by continuous intravenous infusion (IV), for 21 days.
The second patient was a 30 years male ski mountaineer who had suffered a fall over 300 m in open terrain. In addition to the sTBI, he had fractures in the cervical spine, ribs, pelvis, and lower extremities, as well as lung contusions and massive soft tissue trauma. After initial treatment in a local hospital, he was transferred to our department and received the same neuroprotective drugs, like all of our patients with sTBI. Considering the severity of the injuries (Injury Severity Score Rankin Scale, respectively. Currently, both are able to take care of themselves in activities of daily life to a large extent. Neuroprotective drugs may improve the regeneration of cell membranes, improve blood brain barrier integrity, and reduce neuroinflammation leading to secondary damage to the injured brain.

| BACKGROUND
Severe traumatic brain injury (sTBI) is a leading cause of death and disability, especially among young male adults in low-and middle-income countries, shifting to elderly patients in developed countries. This so called "hidden pandemic" affects more than 13 million patients annually in Europe and the US alone. 1 sTBI causes the death of about 11.8/10 5 people in Austria, which is comparable with data from other European countries. 2,3 In survivors, sTBI often has life-changing sequelae such as motor and cognitive deficits, leading to a life of dependency; consequently, sTBI has a significant socio-economic burden and the individual burden. The mechanisms of injury and underlying physiological and cellular processes accompanying the primary and secondary brain injuries are complex and require multimodal treatment. Nevertheless, current guidelines, for example, from the Brain Trauma Foundation (BTF), aim to provide evidence-based recommendations for the various manifestations of sTBI. 4,5 Outcomes may be improved by adequate early care on scene, in the emergency department (ED), 6 and in the intensive care unit (ICU). In addition, sTBI should not be understood as purely acute or static, but as a complex, acute to chronic neurodegenerative disease. It no longer seems so clear that sTBI-induced damage can only be treated within a few hours after the trauma, and the possibility of a longer therapeutic window of time for pharmacological intervention is in discussion. 7 For reduction of secondary damage, anti-oxidants, branched-chain amino acids, and ω-3 polyunsaturated fatty acids have shown promising pre-clinical results. 8 However, pharmacological research has not yet identified a "gold standard" to prevent secondary brain damage. Although there are promising data from animal studies, the only positive findings in retrospective 9,10 and prospective "real life" clinical trials 11,12 were achieved with Citicoline and Cerebrolysin. These were hints of reduced mortality and better functional outcome as measured by enhanced Glasgow Outcome Score (GOSE) and modified Rankin Scale (mRS). 13 However, these agents-whether administered orally or intravenously-have not produced significant clinical improvements when administered as single drugs and are discussed critically in large, randomized prospective trials. [13][14][15]

| CASE 1
A 29-year-old Caucasian male patient collided as motorcyclist head-on with a car at about 65 mph. His helmet was broken on impact with the frame of the car's windscreen. Table 1 shows the patient's status on arrival of emergency medical service (EMS), the pattern of injuries as well as the emergency care measures administered. Whole-body CT scan in the ED revealed punctiform cerebral hemorrhages (especially in the left temporal lobe), bilateral serial rib fractures, a left scapular fracture, prehospital-drained pneumothorax on the left, laceration, and atelectasis of the left lower lobe, ventrocranial edge detachment on the 11th and 12th thoracic vertebrae, wedge shaped compression fractures of the 1st and 4th lumbar vertebrae, and multiple fractures in both hip joints. After placement of a probe to measure the intracranial pressure (ICP) in the ED, the patient was admitted to ICU with stable ventilation and hemodynamic parameters (norepinephrine 0.2 μg/kg/ min). Citicoline (3 g/day) was added to the usual standard care. ICP increased repeatedly up to 19 mmHg during the first days, responding to deepening of analgosedation and osmotherapy. Table 2 shows laboratory findings and hemodynamic support in the first 24 h.
The further course was relatively stable with regard to the ICP and receding brain edema; weaning from ventilation was initiated from Day 10. Subsequently, the patient exhibited an extreme vegetative imbalance, so that in addition to the established antisympathetic (carvedilol) and antipsychotic medication (quetiapine, tiapride, and oxazepam), continuous administration of propofol had to be re-established repeatedly. Three weeks after the accident, magnetic resonance tomography (MRT) revealed signs of intracranial axonal injuries and microbleeds (Figure 1), seen as spotted susceptibility artifacts on the anterior temporal lobe and in the frontobasal region (left > right), in the basal ganglia on both sides, as well as in the left posterior border zone and in the white matter on both sides, in the high frontal to occipital regions. In summary, this indicates Our clinical experience and data suggest that the combined administration of Citicoline and Cerebrolysin may contribute to better recovery, without relevant side effects. However, it would be important to validate these results by means of a controlled, prospective study.

K E Y W O R D S
case series, Cerebrolysin, Citicoline, multi-modal therapy, neuroprotection, secondary brain damage, severe TBI multilocular small shear hemorrhages as well as left frontobasal and temporal contusion zones, compatible with a diffuse axonal trauma stage 1. On Day 21, the patient finally underwent stabilization of both femoral necks with interlocking nails. A dilatative tracheostomy was also performed. Somatosensory evoked potentials (SSEPs) on Day 23 revealed a missing central answer on the right-brain side; on the left a central answer was derived with limited accessibility. During the following days, the patient appeared stressed, showed repeated periods of restlessness with sweating and tremors, and moved his extremities in a completely uncontrolled, stretching manner. Electroencephalogram (EEG) on Day 28 showed signs of general diffuse brain dysfunction (moderate to severe) and reduced reactivity to photogenic stimuli, but no typical epileptic potentials.
On Day 31, more or less with very limited hope for recovery, a supportive therapy with Cerebrolysin was initiated-a novel therapeutic approach for us at that time. 10 days later, the patient began to react to verbal stimulation in a targeted manner for the first time.

T A B L E 1 Status and measures on scene
Further improvement followed rapidly; the neurological examination before transfer to open ward on Day 45 states that the patient was now awake and kept eye contact, gaze tracking was easier to the right than to the left, and muscular tone was generally increased. No cooperation was achieved while examining facial muscles, but he showed indicated movement, when prompted, with fingers and forearm. There was a tendency for spontaneous but uncontrolled stretching spasm of the left leg, but no active movement of the lower extremities when requested. One month later, at the time of discharge from hospital (Day 73), the patient presented awake and mobile in the armchair. He was able to clearly follow simple prompts; questions were adequately answered with a nod of the head. He showed signs of a tetraparesis, pronounced on the left, and a grasping reflex on the right.
One year after the accident, and after a series of botulinum toxin treatments in the rehabilitation center, the disabling spastic increase in muscular tone of the right leg had significantly regressed. The patient was safely mobile, using a three-wheeled bicycle. Six months after this, the patient was mobile on his own by public transportation and regular bicycle, and visiting friends and cafes.
The final MRT examination 18 months after the accident ( Figure 2) shows a gliotic scar cicatricial high parietal remaining as sequelae of the axonal trauma, and a small defect on the left temporal lobe in the area of the previously identified contusion. Overall, the patient had substantially improved; in particular, the frontal contusion zone on the left had receded.

| CASE 2
A 30-year-old sportsman fell from a great height (approx. 300 m, Figure 2) during a ski mountaineering trip. Rescue was delayed due to a lack of cell-phone coverage in this remote area, as well as poor weather conditions. The helicopter emergency service (HEMS) arrived approximately 2 h after the accident. Table 1 shows the patient's status on arrival of HEMS, his pattern of injuries and the emergency care measures administered. The patient was admitted at the nearest trauma center (level II) 4 h after the accident. The CT scan revealed cerebral contusions, traumatic subarachnoid (SAH), and subdural hemorrhage (SDH), an unstable fracture of the 4th cervical vertebra with spinal cord injury, a pneumothorax with rib fractures and multiple fractures of the upper and lower extremities. An external fixator was placed on both lower extremities, followed by plating and screwing of the cervical spine and the left hip on the next day. A postoperative CT scan found extensive brain edema and a new ischemic zone in the area of the posterior circulation, compressing the 4th ventricle.
After being rejected by the nearest neurosurgical university hospital because of the evidently hopeless situation, the patient was transferred to our institution (for laboratory findings and hemodynamic support on admission see   Figure 2) on arrival shows edematous swelling of both cerebellar hemispheres and the right occipital lobe with space-occupying effect in the posterior cranial fossa, and a persistent dislocation fracture of the 4th cervical vertebra. An external ventricular drainage (EVD) was placed, followed by suboccipital decompressive craniotomy with dural expansion surgery on the next day, after interdisciplinary discussion of prognosis, supported by deducible SSEPs over both hemispheres. In addition to standard medical care (comprising "optimal handling," deep analgosedation, hemodynamic, and respiratory optimization) the patient received Cerebrolysin and Citicoline. After prolonged weaning, characterized by vegetative disbalances and delayed recovery from analgosedation, EEG yielded severe abnormal EEG curves with general slowing, no distinct focus, nor typical epileptic potentials. On Day 33 after the trauma, the consulting neurologist stated: Contact via blink of an eye possible, the patient can follow this request. During the examination, the evaluation of pain was possible. The patient was able to keep eye contact only for a short time and was able to raise the right corner of the mouth a little if requested. He was not able to stick out his tongue. Upper extremities (UE): left distal increased tone in the sense of spasticity, right rather flaccid. Lower extremities (LE): both legs immobilized, no examination possible.
After 50 more days in the acute rehabilitation process in ICU including replacing the EVD with a ventriculoperitoneal shunt, the patient was transferred to a specialized clinic for neurorehabilitation. At that time, the report of the neurologist reads as follows: The patient was awake, followed simple prompts, speech comprehension appeared to be well preserved, simple linguistic utterances were possible. Mimic muscles appeared bilaterally symmetrical. He was able to lift and hold UE on the same side against gravity on request, degree of strength 3-4, increase in tone right > left. LE: Tone spastically increased on both sides, right > left. Left leg painful when moving the hip, right leg could be lifted on request.

| OUTCOME AND FOLLOW-UP
One year after the accident, the patient was finally discharged from the rehabilitation center, able to move by himself with a rollator, and was able to independently manage the transfer from bed to wheelchair and vice versa. He shows pronounced deficits in short-term memory; long-term memory is well preserved. He was usually in a euthymic mood and was looking forward to further development steps.

| TREATMENT CONCEPT
Immediately after admission to the ICU of our level 1 trauma center (Wiener Neustadt General Hospital, Austria), all patients with sTBI (Glasgow Coma Score [GCS] 3-8, intubated on scene), who are assessed as having a significant chance of survival, receive Cerebrolysin (50 ml/day) and Citicoline (3 g/day), both given intravenously for 21 days, continuously via motor syringe, in addition to all the standard measures according to BTF guidelines. We hypothesize that the combined effects of these two pleiotropic agents due to their anti-inflammatory properties, reconstitution of the blood-brain-barrier, and stimulation of membrane and axonal regeneration can optimize the neurocognitive outcome. Cerebrolysin is a F I G U R E 2 (A, B) Cranial computer tomography (CCT) scan, Case 2: CCT of a 30-year-old male patient, 1 day after the accident (left image) demonstrates extensive edematous swelling within the anterior cerebellar lobes as well as in both posterior temporal and occipital lobes due to extensive hypoperfusion of the posterior circulation. The cerebellar tentorium (right image) is slightly bowed upward and a slight compression of the basal cistern and the IV ventricle is shown; however, liquor flow was patent. standardized, lipid-free, low-molecular weight neuropeptide preparation, which passes the blood-brain-barrier and mimics the actions of endogenous neurotrophic factors. Proteins present in soluble tissue extracts and in the extracellular matrix have been shown to influence the survival and development of cultured neurons. 16 The tissue-extractderived neuropeptides contained in Cerebrolysin promote neurotrophic stimulation (survival and maintaining the phenotype of highly differentiated neuronal cells), neuromodulation (changes in neuronal and synaptic plasticity synapses), 17 and metabolic regulation such as preventing lactic acidosis and increasing the resilience against hypoxic conditions. 9,12 In addition, Cerebrolysin significantly reduces tissue Plasminogen Activator (tPA)-and fibrin-damaged endothelial cell permeability, which is associated with a significant reduction in proinflammatory and procoagulatory activity. 18 Citicoline (Cytidine 5′-diphosphocholine) is chemically identical to the essential endogenous intermediate in the biosynthetic pathway of phosphatidylcholine. It is hydrolyzed to choline and cytidine triphosphate and crosses the blood-brain barrier to be reassembled to Citicoline inside the brain. It mediates neuronal membrane integrity and repair, and it accelerates the reabsorption of cerebral oedema and the restoration of the blood-brain barrier integrity after TBI. 15,19 Citicoline also increases cerebral dopamine and norepinephrine levels, which causes improved hypoxia tolerance. 20

| DISCUSSION
In these case reports, we describe the clinical development of two patients with sTBI, presenting with GCS 3-4 on site. After surgical intervention both required longterm care at the ICU, followed by intensive rehabilitation measures. Motor vehicle accidents and falls from heights resulting from sports activities are typical causes of sTBI in this age group and of high individual and socio-economic impact. It is of enormous importance to give especially young patients every chance for a life that is as free of disabilities as possible and to reintegrate them into their social environment and work process. Recovery of motor functions and cognitive abilities leads to a significant improvement of activities of daily living. The optimal care for sTBI patients must be started at the emergency site and continued through the emergency department (ED) and the early surgical phase, with ICU treatment closely following the BTF guidelines. 4,5 Starting at the earliest possible time, at the latest when the patient is admitted to the ICU, the described supplementary therapeutic concept should complement the standard treatment of sTBI patients. To the best of our knowledge, these are the first cases reporting this specific pharmacological combination in sTBI, resulting in unexpectedly favorable outcomes (Table 3). In the recent literature, we found only one paper reporting a combined administration of Citicoline and Cerebrolysin in moderate TBI, a RCT, with remarkable positive results. 21 There are two aspects that we attribute to the continuous and high-dose administration of neuroregenerative drugs, among all other attempts to optimize the individual recovery of our patients. Firstly, we did not observe the development of critical brain oedema in these patients, neither in the early nor in the later stages, despite the extremely massive trauma in both cases. Stabilization of damaged cell membranes, reduction and/or acceleration of the absorption of brain oedema are well described effects of Citicoline. 22,23 The need for analgosedation and vasopressors was accordingly moderate. This naturally has a positive effect on gastrointestinal motility, which reduces the risk of translocation and bacteremia. Overall, this results in better stability during the most critical phase of the first 10 days after the trauma.
Secondly, we observed a relatively good neurocognitive long-term outcome in these patients in terms of GOSE and mRS, despite the diffuse axonal trauma, typical for massive acceleration or deceleration trauma. After a phase of "vegetative storm," typically a sign of diffuse axonal trauma, the patients developed positive cognitive abilities over the next few weeks. Despite the prolonged rehabilitation (especially in case 2), the patients recovered much better than expected in terms of cognitive competence and mood stability. The latter may be due in particular to the positive effects of Cerebrolysin, as was shown in the meta-analysis by Ghaffarpasand et al. 24 and the recently published "CAPTAIN" trial. 12 Cerebrolysin may stimulate neurotrophic activity and improve neuromodulation and metabolic regulation. 25 Cerebrolysin and Citicoline were safe in toxicological tests, showing no significant systemic side effects. 23 In rare cases (<1/1000) agitation, loss of appetite, dizziness, or pruritus were observed in awake (stroke) patients receiving Cerebrolysin. In a retrospective cohort study among 7769 adult patients, Abbreviations: ISS, injury severity score; RTS, revised trauma score; TRISS, trauma and injury severity score.
Muresanu et al. did not find any difference in adverse effects between Cerebrolysin and placebo controls. 9 Side effects of Citicoline are also very rare and of mild intensity; single cases suffering from headache, diarrhea, elevated liver enzymes, and weight loss were reported.

| LIMITATIONS
Despite our experience of more than a decade with Citicoline, and about 4 years with Cerebrolysin, we are well aware of the limitations of these exemplary observations and the ongoing discussion about the effectivity of both drugs. Nevertheless, our data can be understood as a hint that neuroprotective and neuoregenerative mechanisms may be supported by these two externally supplied pharmacologic substances. The effects of our multimodal treatment concept may be worth testing in larger, ideally prospective, and randomized clinical trials.

AUTHOR CONTRIBUTIONS
HT and GH concepted and designed this paper; CD and IG contributed to acquisition and analysis of data; JK is responsible for radiologic examination and reporting. HT drafted the article; all authors revised it critically for important intellectual content and approved this version to be published. I, Helmut TRIMMEL, as the corresponding author, have the right to grant on behalf of all authors, an exclusive license for contributions from all authors to permit this work if accepted, to be published.