Peripheral Nervous System Lesions Research Articles

Neutral Red Labeling: A Novel Vital Staining Method for Investigating Central and Peripheral Nervous System Lesions.

Multiple sclerosis, neuromyelitis optica, Guillain-Barré syndrome and chronic inflammatory demyelinating polyradiculoneuropathy are representative demyelinating diseases of the central and peripheral nervous system. Remyelination by myelin forming cells is important for functional recovery from the neurological deficits caused in the demyelinating diseases. Lysophosphatidylcholine-induced demyelination in mice is commonly used to identify and study the molecular pathways of demyelination and remyelination. However, detection of focally demyelinated lesions is difficult and usually requires sectioning of demyelinated lesions in tissues for microscopic analysis. In this review, we describe the development and application of a novel vital staining method for labeling demyelinated lesions using intraperitoneal injection of neutral red (NR) dye. NR labeling reduces the time and effort required to search for demyelinated lesions in tissues, and facilitates electron microscopic analysis of myelin structures. NR labeling also has the potential to contribute to the elucidation of pathologies in the central and peripheral nervous system and assist with identification of drug candidates that promote remyelination.

Wrist extensor pathomechanics: implications for tendon and nerve transfer.

Central and peripheral nervous system lesions may disrupt the intricate balance of the prime movers of the wrist. In spasticity, hyperactive wrist flexors create a flexion moment and, if untreated, can lead to flexion contractures. In patients with C6 spinal cord injury and tetraplegia, the posterior interosseus nerve is typically affected by a complex pattern of upper and/or lower motoneuron lesions causing radial deviation of the wrist due to loss of ulnar deviation actuators. In this report, we illustrate severe pathomechanics that may occur even with relatively modest changes in wrist balance. These results illustrate how thorough understanding of muscle-tendon-joint interaction aids in designing tendon and nerve reconstructive surgeries to normalize wrist positions and balance in neuromuscular conditions.

КЛІНІЧНІ ТА ДЕЯКІ ІМУНОЛОГІЧНІ ОСОБЛИВОСТІ ПРОЯВІВ ГЕРПЕСВІРУСНИХ УРАЖЕНЬ НЕРВОВОЇ СИСТЕМИ НА ФОНІ ПОСТКОВІДНОГО СИНДРОМУ

The article presents data from the current literature on the relevance of a serious health problem, problems in the diagnosis of post-COVID syndrome, complications, complex T-cell and cross-talk immune system responses. A detailed analysis of the manifestations of central and peripheral nervous system lesions in herpesvirus infections with post-COVID syndrome is carried out and substantiated by the literature. The manifestations of immune responses and autoimmune changes in COVID-19 are presented. The aim of the study was to investigate the clinical and immunological features of the course of herpesvirus lesions of the nervous system in patients with and without post-COVID syndrome. We examined 96 patients with nervous system lesions of herpesvirus etiology who had a history of COVID-19 (on average 2-2.5 months before admission to the department). Patients who have had COVID are much more likely to have lesions of individual CNS (2.14 times), PTO dysfunction (2.1 times), diffuse neurological symptoms (2 times), mental disorders (1.8 times), sleep disorders (3.5 times) and fever (2.4 times). The authors of the article found that in patients with nervous system lesions against the background of post-COVID syndrome and activation of neurotropic pathogens, headac he, ataxia and autonomic dysfunction, diffuse neurological symptoms and pyramidal insufficiency were most common. The activity of autoantibodies to neuroantigens (myelin basic protein, NSE, S-100, and human general brain antigen) was determined in all patients. In patients with post-COVID syndrome, a statistically significant increase in the level of autoantibodies to myelin basic protein, S100 protein, and neurospecific enolase was detected compared to the control group. These data indicate a more severe course of nervous system lesions in the setting of post-COVID syndrome, which in turn is associated with deeper damage to the structures of the nervous system. Keywords: Long-COVID, nervous system injures, autoimmune response, herpesviruses.

КЛІНІЧНІ ТА ДЕЯКІ ІМУНОЛОГІЧНІ ОСОБЛИВОСТІ ПРОЯВІВ ГЕРПЕСВІРУСНИХ УРАЖЕНЬ НЕРВОВОЇ СИСТЕМИ НА ФОНІ ПОСТКОВІДНОГО СИНДРОМУ

The article p in the diagnosis of post-COVID syndrome, complications, complex T-cell and cross-talk immune system responses. resents data from the current literature on the relevance of a serious health problem, problems A detailed analysis of the manifestations of central and peripheral nervous system lesions in herpesvirus infections with post-COVID syndrome is carried out and substantiated by the literature. The manifestations of immune responses and autoimmune changes in COVID-19 are presented. The aim of the study was to investigate the clinical and immunological features of the course of herpesvirus lesions of the nervous system in patients with and without post-COVID syndrome. We examined 96 patients with nervous system lesions of herpesvirus etiology who had a history of COVID-19 (on average 2–2.5 months before admission to the department). Patients who have had COVID are much more likely to have lesions of individual CNS (2.14 times), PTO dysfunction (2.1 times), diffuse neurological symptoms (2 times), mental disorders (1.8 times), sleep disorders (3.5 times) and fever (2.4 times). The authors of the article found that in patients with nervous system lesions against the background of postCOVID syndrome and activation of neurotropic pathogens, headac he, ataxia and autonomic dysfunction, diffuse neurological symptoms and pyramidal insufficiency were most common. The activity of autoantibodies to neuroantigens (myelin basic protein, NSE, S100, and human general brain antigen) was determined in all patients. In patients with post-COVID syndrome, a statistically significant increase in the level of autoantibodies to myelin basic protein, S100 protein, and neurospecific enolase was detected compared to the control group. These data indicate a more severe course of nervous system lesions in the setting of postCOVID syndrome, which in turn is associated with deeper damage to the structures of the nervous system. Keywords: Long-COVID, nervous system injures, autoimmune response, herpesviruses.

Characteristics of primary Sjögren's syndrome-associated peripheral nervous system lesions.

The aim of this study is to investigate potential risk factors associated with peripheral nervous system lesions in primary Sjögren's syndrome (pSS) through a retrospective analysis of clinical manifestations, examination characteristics, and clinical electrophysiological features. A retrospective case-control study was conducted at Nanfang Hospital, including 108 patients diagnosed with pSS following the criteria revised by the American College of Rheumatology in 2016. The study spanned from January 2015 to October 2020. The patient cohort was divided into two groups, an experimental group (N = 27) consisting of patients with primary Sjögren's syndrome-peripheral nervous system lesions (pSS-PNS), and a control group (N = 81) comprising patients without peripheral neurological impairment, i.e., primary Sjögren's syndrome-non peripheral nervous system lesions (pSS-nPNS). The results showed a significant correlation between immunoglobulin G (IgG), α-Fodrin immunoglobulin G (α-FIgG), platelet counts (PLT), dry mouth and peripheral neuropathy of Sjogren's syndrome (p < 0.01). The research also revealed that α-FIgG (OR 2.03; 95% CI 1.14-3.64), IgG (OR 1.23; 95% CI 1.06-1.42), and PLT (OR 1.01; 95% CI 1.00-1.01) were identified as risk factors for the onset of peripheral neuropathy of Sjogren's syndrome, while dry mouth had a negative correlation (OR 0.08; 95% CI 0.02-0.40). Remarkably, the total risk assessment of the independent variables demonstrated a high AUC (95%CI) of 0.923 (0.861-0.986; p < 0.001), indicating an excellent prediction of pSS-PNS occurrence through the ROC analysis. Additionally, high platelet counts and strong positive anti-SSB antibody titer were found to be risk factors for dual motor and sensory nerve damages among pSS-PNS patients. IgG, α-FIgG, and PLT were identified as independent risk factors for patients with pSS-PNS. The likelihood of peripheral neuropathy appeared to increase in tandem with the elevated levels of above three factors. Interestingly, we found that dry mouth might play a protective role in this context. Our study further noted that both high platelet counts and strong positive anti-SSB antibody titer may be associated with increased risk of both motor and sensory nerve involvement in pSS-PNS patients. These findings have significant implications for both the etiologies and therapeutics of pSS-PNS.

Neurotrophin-3 (NT-3) as a Potential Biomarker of the Peripheral Nervous System Damage Following Breast Cancer Treatment

Damage to the peripheral nervous system (PNS) is a common complication of breast cancer (BC) treatment, with 60 to 80% of breast cancer survivors experiencing symptoms of PNS damage. In the current study, the levels of brain-derived neurotrophic factor (BDNF), galectin-3 (Gal-3), and neurotrophin-3 (NT-3) were measured in the blood serum of BC patients by ELISA as potential biomarkers that might indicate the PNS damage. Sixty-seven patients were enrolled in this multi-center trial and compared to the aged-matched healthy female volunteers (control group) (n = 25). Intergroup comparison of biomarker levels (i.e., Gal-3 and BDNF) did not show significant differences in any of the studied subgroups. However, intriguingly, NT-3 levels were significantly higher in BC patients as compared to healthy volunteers, constituting 14.85 [10.3; 18.0] and 5.74 [4.56; 13.7] pg/mL, respectively (p < 0.001). In conclusion, NT-3 might be employed as a potential biomarker in BC patients with clinical manifestations of PNS damage. However, further studies to validate its correlation to the degree of peripheral nervous system lesions are of high value.

Long-COVID, neurological and cardiovascular disorders

Introduction. After the disease COVID-19 caused by the SARS-CoV-2 virus, many people recover completely, but others have symptoms that persist for weeks, months or even years after the illness, with the number of patients who are worried about the signs of Long-COVID constantly growing.&#x0D; The aim. Study of the features of the epidemiological and clinical course, pathophysiological mechanisms of long-COVID development, based on the study and analysis of modern scientific data and the establishment of causal relationships of damage to the nervous and cardiovascular systems. &#x0D; Materials and methods. The electronic databases Scopus, PubMed, Web of Science, using modern technologies (Obsidian), key terms and words related to Long COVID, distant neurological and cardio-vascular complications have been studied and analyzed.&#x0D; Conclusions. "Long COVID" is conceptualized as a multiorgan disorder with a wide range of clinical signs that may indicate primarily damage to the nervous and cardiovascular systems. Central or peripheral nervous system lesions occur in more than one-third of patients. Cardiovascular complications, including stroke, are the predominant causes of death of people with "long COVID" (40 %). The most frequent neurological signs of "Long COVID" (&gt;70 %) are fatigue, headache, sleep disorders, emotional disorders and sensorimotor disorders. The processes of neuroinflammatory and oxidative stress predominate in the spread of neurological consequences of "Long COVID".

The best of both worlds: mastering nerve regeneration combining biological and nanotechnological tools.

The best of both worlds: mastering nerve regeneration combining biological and nanotechnological tools.

Single-Center Retrospective Analysis of Paraneoplastic Syndromes with Peripheral Nerve Damage.

There are few clinical and electrophysiological studies on paraneoplastic neurological syndrome (PNS) with peripheral nerve damage, which brings great challenges to clinical identification and diagnosis. We analyzed the clinical and electrophysiological data of twenty-five confirmed PNS cases using peripheral nerve damage patients. The results showed the most common chief complaint was weakness (20/25, 80%), followed by numbness (13/25, 52%). Nineteen patients (76%) exhibited peripheral nervous system lesions prior to occult tumors, and the median time from symptom onset to the diagnosis of a tumor was 4 months. The electrophysiological results revealed a higher rate of abnormal amplitudes than latency or conduction velocity, especially in sensory nerves. Meanwhile, we found that, compared with patients &gt;65 y, patients aged ≤65 y exhibited more chronic onset (p = 0.01) and longer disease duration (p = 0.01), more motor nerve involvements (p = 0.02), more amplitude involvement (p = 0.01), and higher rates of the inability to walk independently at presentation (p = 0.02). The present study construed that weakness and paresthesia are common symptoms in PNS with peripheral nerve damage in some areas, and the electrophysiological results mainly changed in amplitude. Tumor screening in young and middle-aged patients with peripheral neuropathy cannot be ignored.

The correlation between the body shape and otolithic function in patients with obstructive sleep apnea

ObjectiveTo identify the typical pattern of changes of vestibular-evoked myogenic potentials (VEMPs) and explore the relationship between VEMPs and the anthropometry factors in patients with obstructive sleep apnea (OSA). MethodsPatients diagnosed as OSA after overnight polysomnography (PSG) tests were enrolled as the study group. Healthy volunteers were recruited as the control group. Anthropometry data of the body shape and VEMPs results were collected completely. The correlation analysis was conducted among those parameters. ResultsForty-nine patients with OSA who were diagnosed in the Therapy Center of Sleep-disordered Breathing in our hospital and sex- and age-matched healthy controls as well. Significant changes in ocular and cervical VEMPs (oVEMP and cVEMP) in the study group were observed, which were reduced response rates, elevated thresholds, decreased amplitudes, and prolonged first wave latencies. In oVEMP, the first wave (n1) latency was significantly correlated with weight, body mass index (BMI), neck circumference, waist circumference, hip circumference, and apnea hypopnea index (AHI). In a tentative application, combined use of BMI and oVEMP n1 latency increased the detection rate during OSA screening prior to PSG. ConclusionOSA can negatively affect function of otolithic organs and their pathways. The first wave latency of the VEMPs waveform may be another important parameter to define peripheral nervous system lesions caused by systemic diseases as OSA.

Transplantation of Olfactory Ensheathing Cells: Properties and Therapeutic Effects after Transplantation into the Lesioned Nervous System

The primary olfactory system (POS) is in permanent renewal, especially the primary olfactory neurons (PON) are renewed with a turnover of around four weeks, even in adulthood. The re-growth of these axons is helped by a specific population of glial cells: the olfactory ensheathing cells (OECs). In the POS, OECs constitute an “open-channel” in which the axons of PON cause regrowth from peripheral nervous system (PNS) to central nervous system (CNS). The remarkable role played by OECs into the POS has led scientists to investigate their properties and potential beneficial effects after transplantation in different lesion models of the CNS and PNS. In this review, we will resume and discuss more than thirty years of research regarding OEC studies. Indeed, after discussing the embryonic origins of OECs, we will describe the in vitro and in vivo properties exert at physiological state by these cells. Thereafter, we will present and talk over the effects of the transplantation of OECs after spinal cord injury, peripheral injury and other CNS injury models such as demyelinating diseases or traumatic brain injury. Finally, the mechanisms exerted by OECs in these different CNS and PNS lesion paradigms will be stated and we will conclude by presenting the innovations and future directions which can be considered to improve OECs properties and allow us to envisage their use in the near future in clinical applications.

Bladder cancer in patients with neurogenic bladder disorder: a comparative study of different etiologies.

The objective of this study was to conduct a comparative study of different etiologies of neurogenic bladder disorders (NBDs) in patients with bladder cancer (BC) regarding patient- and tumor-related parameters and their oncological outcome. Out of 98 patients with bladder tumors and neurogenic disease, 23 patients with BC and NBDs from Jan 1, 2010, to Dec 31, 2020, were included. The different etiologies of NBDs were merged in three groups based on the level of the nervous system (NS)-lesion: (i) central (n = 6), (ii) spinal cord (n = 10), and (iii) peripheral lesions (n = 7). Patients with lesions at the spinal cord level were younger at the time of BC diagnosis compared to patients with central or peripheral NS lesions (54 vs. 68 vs. 78years, p = 0.0219). However, the latency to malignant transformation was more than twice as long (33 vs. 15years, p = 0.0108). Most tumors were muscle-invasive or locally advanced BCs (62%) and presented lymph node metastases (55%), resulting in a poor mean overall survival of 30.9 ± 3.6months. No significant differences regarding histopathology, tumor stage, and oncological outcome could be observed between the groups. Patients with NBDs have a poor prognosis regardless of their etiology or the level of NS lesion. Patients with spinal cord lesions, including congenital NBDs, appear to develop BC at a young age, but compared to other etiologies latency from NBD to BC is longer.

Electrodiagnostic evaluation in differential diagnosis and neurosurgical treatment of radiculopathies caused by spine disorders. Diagnostic value and methodology

To assess the role of electromyography including paraspinal muscle mapping in diagnosis of radiculopathies following spine diseases. We have analyzed literature data in the Scopus, Pubmed, and RSCI databases and selected 93 references for primary reviewing. Final analysis enrolled the manuscripts with a detailed description of neurophysiological examinations and data on sensitivity/specificity of these methods. Needle electromyography (EMG) is the most informative neurophysiological method for diagnosis of radicular damage. Sensitivity of EMG is up to 90% for lumbosacral radiculopathy. Electromyography of the paraspinal muscles can be used in case of of cervical, thoracic and lumbar radiculopathy in addition to EMG of limb muscles. Therefore sensitivity increases to 100%. Diagnostic value of nerve conduction study (NCS) is low, and performing NCS without EMG is not useful. In neurosurgical practice electrodiagnostic (EDX) should be performed for differential diagnosis of radiculopathy and peripheral nervous system lesions, to determine the level of radicular compression, and when physical examination does not correspond with neuroimaging or MRI is not possible to perform.

Macroscopic detection of demyelinated lesions in mouse PNS with neutral red dye

Lysophosphatidylcholine (LPC)-induced demyelination is a versatile animal model that is frequently used to identify and examine molecular pathways of demyelination and remyelination in the central (CNS) and peripheral nervous system (PNS). However, identification of focally demyelinated lesion had been difficult and usually required tissue fixation, sectioning and histological analysis. Recently, a method for labeling and identification of demyelinated lesions in the CNS by intraperitoneal injection of neutral red (NR) dye was developed. However, it remained unknown whether NR can be used to label demyelinated lesions in PNS. In this study, we generated LPC-induced demyelination in sciatic nerve of mice, and demonstrated that the demyelinated lesions at the site of LPC injection were readily detectable at 7 days postlesion (dpl) by macroscopic observation of NR labeling. Moreover, NR staining gradually decreased from 7 to 21 dpl over the course of remyelination. Electron microscopy analysis of NR-labeled sciatic nerves at 7 dpl confirmed demyelination and myelin debris in lesions. Furthermore, fluorescence microscopy showed NR co-labeling with activated macrophages and Schwann cells in the PNS lesions. Together, NR labeling is a straightforward method that allows the macroscopic detection of demyelinated lesions in sciatic nerves after LPC injection.

Neuroimaging Diagnosis of Prefrontal Cortex and Peripheral Nervous System Lesions in Patients With Bipolar Disorder

双向障碍；前额叶皮层；边缘神经系统；结构影像学；功能影像学

Comparative Pathology of the Peripheral Nervous System.

The peripheral nervous system (PNS) relays messages between the central nervous system (brain and spinal cord) and the body. Despite this critical role and widespread distribution, the PNS is often overlooked when investigating disease in diagnostic and experimental pathology. This review highlights key features of neuroanatomy and physiology of the somatic and autonomic PNS, and appropriate PNS sampling and processing techniques. The review considers major classes of PNS lesions including neuronopathy, axonopathy, and myelinopathy, and major categories of PNS disease including toxic, metabolic, and paraneoplastic neuropathies; infectious and inflammatory diseases; and neoplasms. This review describes a broad range of common PNS lesions and their diagnostic criteria and provides many useful references for pathologists who perform PNS evaluations as a regular or occasional task in their comparative pathology practice.

FEATURES OF CLINICAL MANIFESTATIONS OF POST-TRAUMATIC NEUROPATHIES AND PLEXOPATHIES RESULTED FROM GUNSHOT AND NON-GUNSHOT INJURIES OF EXTREMITIES

The number of traumatic injuries to nerve stems and plexuses is steadily increasing in peacetime during armed conflicts and terrorist acts. In wartime, peripheral nerve injuries are much more common, and a great deal of the knowledge about peripheral nerve damage and repair is based on combat experience. The study of the clinical course of post−traumatic neuropathies and plexopathies contributes to the development of clinical and neurological criteria and compensatory−restorative responses in traumatic lesions of the peripheral nervous system, helps to assess the functional significance of various parts of the nervous system when compensating a damaged functional unit. To study the features of clinical manifestations of post−traumatic gunshot and non−gunshot neuropathies and plexopathies, 63 patients underwent clinical and neurological examination with topical and clinical diagnoses, collection of detailed anamnesis and complaints, electroneuromyography and ultrasound examination. Movement disorders, characterized by peripheral paresis or plegia of the corresponding muscle group and accompanied with a reduced or lost tendon and periosteal reflexes, were common. Sensitivity disorders were a combination of prolapse (anesthesia, hypoesthesia) and irritation (paresthesia, hyperpathy, hyperesthesia). Autonomic disorders (vascular, secretory and trophic) in traumatic neuropathies differ depending on the clinical individuality of peripheral nerves. Vascular disorders were more often detected with partial damage to nerve structures and were accompanied by local edema. Of the secretory disorders, the most constant sign of impaired nerve conduction was sweating disorder. In the clinical picture of the pain syndrome, i.e. causalgia, the pain sensations by type of burning dominated. The intensity of the pain syndrome in severe cases was very high, in some cases the pain was exacerbated by irritation of the senses. The clinical picture of causalgia is characterized by an increased pain when warming the injured limb and it reduced when cooled, that is a "symptom of a wet rag." Knowledge of clinical features allows the detection of the peripheral nervous system lesions at the early stages of pathology, performance of dynamic clinical and neurological observation and treatment, timely use of modern additional research methods to address further treatment tactics that restore limb function, improve quality of life. Key words: post−traumatic neuropathy and plexopathy, peripheral nervous system, gunshot wounds of nerves and plexuses.

Intercellular communication and ion channels in neuropathic pain chronicization.

Neuropathic pain is caused by primary lesion or dysfunction of either peripheral or central nervous system. Due to its complex pathogenesis, often related to a number of comorbidities, such as cancer, neurodegenerative and neurovascular diseases, neuropathic pain still represents an unmet clinical need, lacking long-term effective treatment and complex case-by-case approach. We analyzed the recent literature on the role of selective voltage-sensitive sodium, calcium and potassium permeable channels and non-selective gap junctions (GJs) and hemichannels (HCs) in establishing and maintaining chronic neuropathic conditions. We finally focussed our review on the role of extracellular microenvironment modifications induced by resident glial cells and on the recent advances in cell-to-cell and cell-to-extracellular environment communication in chronic neuropathies. In this review, we provide an update on the current knowledge of neuropathy chronicization processes with a focus on both neuronal and glial ion channels, as well as on channel-mediated intercellular communication.

«Точка невозврата» при диабетических невропатиях: заблуждение или преступление? Международный консенсус

Polyneuropathy in patients with diabetes mellitus is manifested by a lesion of peripheral sensory, motor and autonomic nervous system. Different severity of damage of sensory, motor and autonomic fibers in typical and atypical forms of diabetic polyneuropathy, require a differentiated approach to therapy, but not the rejection of its implementation. In an interdisciplinary consensus, consultations are held with physicians from different regions of the Russian Federation, and modern methods of diagnosing and assessing the severity of diabetic polyneuropathies, which determine the algorithm for treating patients, are discussed. Keywords: diabetic polyneuropathy, neuropathic pain, gabapentinoids, antidepressants, thioctic acid, benfotiamine, neurotropic therapy, pentasinergy, neuroplasticity, point of no return, therapeutic nihilism, interdisciplinary consensus on DPN.

Peripheral nervous system lesion in systemic vasculitis - issues of diagnosis and treatment

Vasculitis is a clinically diverse group of diseases with histopathological signs of blood vessel inflammation, which contributes to vascular damage and ischemic damage to the affected tissues. Vasculitic neuropathy is a common complication of the primary systemic vasculitides, such as polyartertis nodosa and antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, systemic diseases of the connective tissue - systemic lupus erythematosus and Sjogren syndrome, vasculitis associated with infection, most often viral hepatitis C and B and non - systemic vasculitis neuropathy. Vessels of medium and small caliber are involved in the pathological process in these diseases. With all vasculitis, except for those caused by the direct effect of the infectious trigger on the blood vessel walls, the main pathogenetic mechanism is an autoimmune process with the development of vasa nervorum vasculitis - small arteries and vessels that supply peripheral nerves, and the outcome - nerve ischemia. The classic clinical presentation is an acute or subacute painful multifocal neuropathy that has a predilection for the lower extremities, affects two or more named nerves, and progresses in a step wise manner. However, vasculitic neuropathy can manifest in a variety of ways, including asymmetric polyneuropathies and distal symmetric sensory neuropathies, and it also can be slowly progressive, particularly in cases of nonsystemic vasculitic neuropathy (NSVN), a form of vasculitis that clinically remains restricted to peripheral nerves. Nerve biopsy can help establish the diagnosis of a systemic vasculitis, particularly when other organ involvement is not clinically apparent, and is required for diagnosis of NSVN. Neuropathy due to systemic vasculitis should be treated in accordance with the recommendations for the treatment of the underlying disease. In NSVH, the main medicine of choice are glucocrticoids, and in severe/progressive cases, pulse therapy with cyclophosphamide.