Brain washing and neural health: role of age, sleep, and the cerebrospinal fluid melatonin rhythm.

The brain lacks a classic lymphatic drainage system. How it is cleansed of damaged proteins, cellular debris, and molecular by-products has remained a mystery for decades. Recent discoveries have identified a hybrid system that includes cerebrospinal fluid (CSF)-filled perivascular spaces and classic lymph vessels in the dural covering of the brain and spinal cord that functionally cooperate to remove toxic and non-functional trash from the brain. These two components functioning together are referred to as the glymphatic system. We propose that the high levels of melatonin secreted by the pineal gland directly into the CSF play a role in flushing pathological molecules such as amyloid-ß peptide (Aß) from the brain via this network. Melatonin is a sleep-promoting agent, with waste clearance from the CNS being highest especially during slow wave sleep. Melatonin is also a potent and versatile antioxidant that prevents neural accumulation of oxidatively-damaged molecules which contribute to neurological decline. Due to its feedback actions on the suprachiasmatic nucleus, CSF melatonin rhythm functions to maintain optimal circadian rhythmicity, which is also critical for preserving neurocognitive health. Melatonin levels drop dramatically in the frail aged, potentially contributing to neurological failure and dementia. Melatonin supplementation in animal models of Alzheimer's disease (AD) defers Aß accumulation, enhances its clearance from the CNS, and prolongs animal survival. In AD patients, preliminary data show that melatonin use reduces neurobehavioral signs such as sundowning. Finally, melatonin controls the mitotic activity of neural stem cells in the subventricular zone, suggesting its involvement in neuronal renewal.

Melatonin modulates the Warburg effect and alters the morphology of hepatocellular carcinoma cell line resulting in reduced viability and migratory potential.

AIMS: Hepatocellular Carcinoma (HCC) is a primary neoplasm derived from hepatocytes with low responsiveness and recurrent chemoresistance. Melatonin is an alternative agent that may be helpful in treating HCC. We aimed to study in HuH 7.5 cells whether melatonin treatment exerts antitumor effects and, if so, what cellular responses are induced and involved. MAIN METHODS: We evaluated the effects of melatonin on cell cytotoxicity and proliferation, colony formation, morphological and immunohistochemical aspects, and on glucose consumption and lactate release. KEY FINDINGS: Melatonin reduced cell motility and caused lamellar breakdown, membrane damage, and reduction in microvillus. Immunofluorescence analysis revealed that melatonin reduced TGF and N-cadherin expression, which was further associated with inhibition of epithelial-mesenchymal transition process. In relation to the Warburg-type metabolism, melatonin reduced glucose uptake and lactate production by modulating intracellular lactate dehydrogenase activity. SIGNIFICANCE: Our results indicate that melatonin can act upon pyruvate/lactate metabolism, preventing the Warburg effect, which may reflect in the cell architecture. We demonstrated the direct cytotoxic and antiproliferative effect of melatonin on the HuH 7.5 cell line, and suggest that melatonin is a promising candidate to be further tested as an adjuvant to antitumor drugs for HCC treatment.

Melatonin Reverses the Warburg-Type Metabolism and Reduces Mitochondrial Membrane Potential of Ovarian Cancer Cells Independent of MT1 Receptor Activation.

Ovarian cancer (OC) is the most lethal gynecologic malignancy, and melatonin has shown various antitumor properties. Herein, we investigated the influence of melatonin therapy on energy metabolism and mitochondrial integrity in SKOV-3 cells and tested whether its effects depended on MT1 receptor activation. SKOV-3 cells were exposed to different melatonin concentrations, and experimental groups were divided as to the presence of MT1 receptors (melatonin groups) or receptor absence by RNAi silencing (siRNA MT1+melatonin). Intracellular melatonin levels increased after treatment with melatonin independent of the MT1. The mitochondrial membrane potential of SKOV-3 cells decreased in the group treated with the highest melatonin concentration. Melatonin reduced cellular glucose consumption, while MT1 knockdown increased its consumption. Interconversion of lactate to pyruvate increased after treatment with melatonin and was remarkable in siRNA MT1 groups. Moreover, lactate dehydrogenase activity decreased with melatonin and increased after MT1 silencing at all concentrations. The UCSC XenaBrowser tool showed a positive correlation between the human ASMTL gene and the ATP synthase genes, succinate dehydrogenase gene (SDHD), and pyruvate dehydrogenase genes (PDHA and PDHB). We conclude that melatonin changes the glycolytic phenotype and mitochondrial integrity of SKOV-3 cells independent of the MT1 receptor, thus decreasing the survival advantage of OC cells.

Melatonin: A mitochondrial resident with a diverse skill set.

Melatonin is an ancient molecule that originated in bacteria. When these prokaryotes were phagocytized by early eukaryotes, they eventually developed into mitochondria and chloroplasts. These new organelles retained the melatonin synthetic capacity of their forerunners such that all present-day animal and plant cells may produce melatonin in their mitochondria and chloroplasts. Melatonin concentrations are higher in mitochondria than in other subcellular compartments. Isolated mouse oocyte mitochondria form melatonin when they are incubated with serotonin, a necessary precursor. Oocyte mitochondria subsequently give rise to these organelles in all adult vertebrate cells where they continue to synthesize melatonin. The enzymes that convert serotonin to melatonin, i.e., arylalkylamine-N-acetyltransferase (AANAT) and acetylserotonin-O-methyltransferase, have been identified in brain mitochondria which, when incubated with serotonin, also form melatonin. Melatonin is a potent antioxidant and anti-cancer agent and is optimally positioned in mitochondria to aid in the maintenance of oxidative homeostasis and to reduce cancer cell transformation. Melatonin stimulates the transfer of mitochondria from healthy cells to damaged cells via tunneling nanotubes. Melatonin also regulates the major NAD+-dependent deacetylase, sirtuin 3, in the mitochondria. Disruptions of mitochondrial melatonin synthesis may contribute to a number of mitochondria-related diseases, as discussed in this review.

The proteomic landscape of ovarian cancer cells in response to melatonin.

Ovarian cancer (OC) is the most lethal gynecological malignancy with a highly negative prognosis. Melatonin is an indoleamine secreted by the pineal gland during darkness and has shown antitumor activity in both in vitro and in vivo experiments. Herein, we investigated the influence of melatonin on the proteome of human ovarian carcinoma cells (SKOV-3 cell line) using the Ultimate 3000 LC Liquid NanoChromatography equipment coupled to a Q-Exactive mass spectrometry. After 48 h of treatment, melatonin induced a significant cytotoxicity especially with the highest melatonin concentration. The proteomic profile revealed 639 proteins in the control group, and 98, 110, and 128 proteins were altered by melatonin at the doses of 0.8, 1.6, and 2.4 mM, respectively. Proteins associated with the immune system and tricarboxylic acid cycle were increased in the three melatonin-exposed groups of cells. Specifically, the dose of 2.4 mM led to a reduction in molecules associated with protein synthesis, especially those of the ribosomal protein family. We also identified 28 potential genes shared between normal ovarian tissue and OC in all experimental groups, and melatonin was predicted to alter genes encoding ribosomal proteins. Notably, the set of proteins changed by melatonin was linked to a better prognosis for OC patients. We conclude that melatonin significantly alters the proteome of SKOV-3 cells by changing proteins involved with the immune response and mitochondrial metabolism. The concentration of 2.4 mM of melatonin promoted the largest number of protein changes. The evidence suggests that melatonin may be an effective therapeutic strategy against OC.

Caffeine consumption attenuates ethanol-induced inflammation through the regulation of adenosinergic receptors in the UChB rats cerebellum.

Caffeine consumption is able to interfere in cellular processes related to inflammatory mechanisms by acting through the adenosinergic system. This study aimed to recognize alterations related to adenosinergic system and inflammatory process in the cerebellum of University of Chile Bibulous (UChB) rats after the consumption of ethanol and caffeine. UChB and Wistar rats, males at 5 months old, were divided into the groups (n = 15/group): (i) Control (Wistar rats receiving water); (ii) Ethanol group (UChB rats receiving ethanol solution at 10%) and (iii) Ethanol+caffeine group (UChB rats receiving ethanol solution at 10% added of 3 g/L of caffeine). The cerebellar tissue was collected and processed for immunohistochemistry, Reverse transcription polymerase chain reaction (RT-PCR) and western blotting techniques for the adenosinergic receptors A1 and A2a and inflammatory markers, including Nuclear factor kappa B (NFkB), TLR4, TLR2, MyD88, TNF-α, COX-2, iNOS and microglial marker Iba-1. Results showed ethanol and caffeine consumption differentially altering the immunolocalization of adenosinergic receptors and inflammatory markers in the cerebellar tissue. The A2a receptor was overexpressed in the Ethanol group and was evident in the glial cells. The Ethanol group had increased protein levels for NFκB and TLR4, expressively in Bergmann glia and Purkinje cells. Caffeine reduced the expression of these markers to levels similar to those found in the Control group. The A1 gene was upregulated the Ethanol group, but not its protein levels, suggesting post-transcriptional interference. In conclusion, caffeine seems to attenuate ethanol-induced inflammation in the cerebellum of UChB rats through the A1 and A2a modulation, playing a neuroprotective role in the chronic context of ethanol consumption.

Nandrolone decanoate causes uterine injury by changing hormone levels and sex steroid receptors in a dose- and time-dependent manner.

Different doses of nandrolone decanoate (ND) were used to investigate the expression of uterine sex steroid receptors (AR, ER-α, and ER-ß) and the levels of serum sex hormones after treatment and recovery periods in adult rats. ND doses of 1.87, 3.75, 7.5, or 15 mg/kg b.w. or mineral oil (control group) were injected subcutaneously for 15 days, and the experimental groups were divided into three periods of evaluation: (a) ND treatment for 15 days, (b) ND treatment followed by 30-day-recovery and (c) ND treatment followed by 60-day-recovery. Estrous cycle was monitored daily. At the end of each experimental period, rats were euthanized for the collection of serum samples and uterine tissues. All animals showed persistent diestrus and only the highest ND dose was capable of inducing persistent diestrus until 60-day-recovery. Immunoexpression of uterine sex steroid receptors varied in a time-dependent manner. While AR expression was increase after treatment period, ER-α and ER-ß expressions decreased after 60- and 30-day-recovery, respectively. ND also increased the serum levels of testosterone, 17ß-estradiol, and dihydrotestosterone, especially at the highest doses of 7.5 and 15 mg ND/kg until 30 days of recovery. The levels of progesterone were significantly reduced in all ND-treated animals. No significant difference was observed in the levels of follicle-stimulating hormone, whereas the levels of luteinizing hormone varied according to specific dose and period. We conclude that uterine sex steroid receptors and sex hormones are affected by ND administration and these alterations can be only restored following lower doses and long recovery periods.

Pterostilbene influences glycemia and lipidemia and enhances antioxidant status in the liver of rats that consumed sucrose solution.

AIMS: The present study investigated the potential effects of pterostilbene (PT) on glycemic and lipid profiles, fat storage, cardiovascular indices, and hepatic parameters of rats fed with sucrose solution. MAIN METHODS: 24 male Wistar rats received either drinking water or a 40% sucrose solution over a period of 140 days. After this period, animals were randomly allocated into four groups (n = 6): Control (C), C + Pterostilbene (PT), Sucrose (S), and S + PT. Pterostilbene (40 mg/kg) was given orally for 45 consecutive days. KEY FINDINGS: Pterostilbene did not influence morphometric and nutritional parameters. The insulin sensitivity index TyG was elevated in the C + PT group (p < 0.01) and reduced in S + PT group (p < 0.05). Basal glucose levels were lower in the S + PT group (p < 0.05), and the glycemic response was improved with PT treatment in glucose provocative tests. Conversely, rats from the C + PT group showed impaired glucose disposal during those tests. Lipid profile was partially improved by PT treatment. Hepatic oxidative stress in the S group was improved after PT treatment. In the C group, PT reduced SOD activity, glutathione levels, and increased catalase activity. Collagen content was reduced by PT treatment. SIGNIFICANCE: PT effects depends on the type of diet the animals were submitted. In rats fed with sucrose-solution, PT confirmed its positive effects, improving glucose and lipid profile, and acting as a potent antioxidant. The effects of PT on rats that consumed a normal diet were very discrete or even undesirable. We suggest caution with indiscriminate consume of natural compounds by healthy subjects.

Maternal protein restriction impairs nutrition and ovarian histomorphometry without changing p38MAPK and PI3K-AKT-mTOR signaling in adult rat ovaries.

AIMS: Because an adequate protein supply is detrimental for the maintenance of folliculogenesis and ovulation, we evaluated the impact of maternal low protein diet on nutritional parameters, estrous cycle, ovarian histomorphometry, and on the expression of metabolic and survival signaling molecules in different follicular stages. MAIN METHODS: Twenty Wistar pregnant rats were divided into two groups: the normoprotein (NP) group, composed of animals that received 17% protein, and a low-protein (LP) group, composed of animals that received 6% protein during gestation and lactation period. After weaning, female rats were fed with standard diet until the 120-days-old. KEY FINDINGS: LP animals showed reduced body mass index, total body weight, energy intake, feed efficiency, and visceral fat. The ovarian tissue presented vascular congestion and fat accumulation in the medulla, followed by a significant reduction in the amount of primordial and primary follicles. In addition, the number of atretic follicles was higher in LP than in NP animals. Maternal undernutrition also resulted in increased levels of estradiol (E2) and progesterone (P4) while testosterone (T) was unchanged in the offspring. Although discrete changes in p38MAPK and in PI3K-AKT-mTOR immunostaining were observed in the ovarian follicles and corpus luteum in LP, no differences were found at their protein levels. SIGNIFICANCE: Maternal protein restriction alters estrous cycle and histomorphometry of the offspring's ovary without changing the levels of intracellular regulatory molecules in adulthood. These morphofunctional changes may alter reproductive performance in female offspring, highlighting maternal dietary conditions as an important factor for offspring reproductive health.

P-MAPA activates TLR2 and TLR4 signaling while its combination with IL-12 stimulates CD4+ and CD8+ effector T cells in ovarian cancer.

AIMS: Ovarian cancer (OC) is the most lethal gynecological malignancies and many women develop chemoresistance associated with the inflammatory process. We investigated the effects of P-MAPA and IL-12 on the inflammatory and immune responses in a chemically-induced OC model. MAIN METHODS: OCs were induced with 7,12-dimethylbenz(a)anthracene into the ovarian bursa, and the animals were given P-MAPA (5 mg/kg bw., i.p., twice a week), or IL-12 (300 ng/kg bw., i.p., one a week) for 60 days, or both P-MAPA and IL-12. Immunohistochemistry, western blot, flow cytometry, and multiplex assay were used to examine the effectiveness of immunotherapies in OC. KEY FINDINGS: The combinatory therapy improved the general OC features, reducing inflammatory cells and adipocyte accumulation, in addition to revealing a soft and mobile tissue with no adherences and peritoneal implants. P-MAPA treatment increased the levels of TLR2, TLR4 and TRIF in OCs while decreasing the number of regulatory T (Treg) cells. Additionally, the association of P-MAPA with IL-12 significantly increased the number of CD4+ and CD8+ T effector cells in draining lymph nodes. Regarding the inflammatory mediators, P-MAPA enhanced the levels of the pro-inflammatory cytokine IL-17 while P-MAPA+IL-12 increased the levels of IL-1ß. Treatment with IL-12 enhanced the cytokine levels of IL-17, TNF-α, IL-1ß, and IL-2 in addition to the chemokine MIP-1α. SIGNIFICANCE: We conclude that P-MAPA upregulated TLR2 and TLR4 signaling, possibly activating the non-canonical pathway, while attenuating the tumor immunosuppression. Also, the combination of P-MAPA with IL-12 improves the antitumor immunoresponse, opening a new therapeutic approach for fighting OC.

The role of Toll-like receptor 4 signaling pathway in ovarian, cervical, and endometrial cancers.

Toll-like receptors (TLRs) are critical sensors related to inflammation and tumorigenesis. Among all subtypes, the TLR4 is a highly described transmembrane protein involved in the inflammatory process. The TLR4/myeloid differentiation factor 88 (MyD88) signaling pathway has been implicated in oncogenic events in several tissues and is associated with survival of patients. Through activation, TLR4 recruits adaptor proteins, i.e., MyD88 or TRIF, to triggers canonical and non-canonical signaling pathways that result in distinct immune responses. In most cancer cells, uncontrolled TLR4 signaling modifies the tumor microenvironment to proliferate and evade immune surveillance. By contrast, TLR4 activation can produce antitumor activities, thereby inhibiting tumor growth and enhancing the proper immune response. We review herein recent approaches on the role of the TLR4 signaling pathway and discuss potential candidates for gynecological cancer therapies; among these agents, natural and synthetic compounds have been tested both in vitro and in vivo. Since TLR4 ligands have been investigated as effective immune-adjuvants in the context of these aggressive malignancies, we described how TLR4 signaling controls part of the tumor-related inflammatory process and which are the new targeting molecules implicated in the regulation of tumorigenicity in ovarian, cervical, and endometrial cancers.

Physical resistance training-induced changes in lipids metabolism pathways and apoptosis in prostate.

BACKGROUND: Altered lipid metabolism is an important characteristic of neoplastic cells, with androgens and growth factors being major regulatory agents of the lipid metabolism process. We investigated the effect of physical resistance training on lipid metabolism and apoptosis in the adult Wistar rat prostate. METHODS: Two experimental groups represented sedentary and physical resistance training. Three days per week for 13 weeks, rats performed jumps in water carrying a weight load strapped to their chests as part of a physical resistance exercise protocol. Two days after the last training session, rats were anesthetized and sacrificed for blood and prostate analysis. RESULTS: Physical exercise improved feeding efficiency, decreased weight gain, regulated the serum-lipid profile, and modulated insulin-like growth factor-1 (IGF-1) and free testosterone concentration. Furthermore, upregulation of cluster of differentiation 36 (CD36), sterol regulatory element binding protein-1 (SREBP-1), sterol regulatory element-binding protein cleavage-activating protein (SCAP), and reduced lysosome membrane protein (LIMPII) expression were also observed in the blood and prostates of trained rats. Consistent with these results, caspase-3 expression was upregulating and the BCL-2/Bax index ratio was decreased in trained rats relative to sedentary animals. CONCLUSIONS: In this work, physical resistance training can alter lipid metabolism and increase markers of apoptosis in the prostate, suggesting physical resistance training as a potential novel therapeutic strategy for treating prostate cancer.

Mitochondrial functions and melatonin: a tour of the reproductive cancers.

Cancers of the reproductive organs have a strong association with mitochondrial defects, and a deeper understanding of the role of this organelle in preneoplastic-neoplastic changes is important to determine the appropriate therapeutic intervention. Mitochondria are involved in events during cancer development, including metabolic and oxidative status, acquisition of metastatic potential, resistance to chemotherapy, apoptosis, and others. Because of their origin from melatonin-producing bacteria, mitochondria are speculated to produce melatonin and its derivatives at high levels; in addition, exogenously administered melatonin accumulates in the mitochondria against a concentration gradient. Melatonin is transported into tumor cell by GLUT/SLC2A and/or by the PEPT1/2 transporters, and plays beneficial roles in mitochondrial homeostasis, such as influencing oxidative phosphorylation and electron flux, ATP synthesis, bioenergetics, calcium influx, and mitochondrial permeability transition pore. Moreover, melatonin promotes mitochondrial homeostasis by regulating nuclear DNA and mtDNA transcriptional activities. This review focuses on the main functions of melatonin on mitochondrial processes, and reviews from a mechanistic standpoint, how mitochondrial crosstalk evolved in ovarian, endometrial, cervical, breast, and prostate cancers relative to melatonin's known actions. We put emphasis on signaling pathways whereby melatonin interferes within cancer-cell mitochondria after its administration. Depending on subtype and intratumor metabolic heterogeneity, melatonin seems to be helpful in promoting apoptosis, anti-proliferation, pro-oxidation, metabolic shifting, inhibiting neovasculogenesis and controlling inflammation, and restoration of chemosensitivity. This results in attenuation of development, progression, and metastatic potential of reproductive cancers, in addition to lowering the risk of recurrence and improving the life quality of patients.

Histopathological changes in androgenized ovaries are recovered by melatonin treatment.

Nandrolone decanoate (ND) is a synthetic steroid, which promotes adverse effects on the ovarian tissue, and melatonin (MLT) exhibits a number of beneficial properties in the reproductive system. This study evaluated the general features of the ovarian tissue and the immunoexpression of sex steroid receptors in ND-treated rats that were submitted to short-term melatonin treatment. Adult rats received mineral oil (control group) and ND at doses of 7.5 mg/kg for 15 days (ND-treated group). The treatment with MLT (10mg/kg for 7 days) was given alone, before or in combination with ND. All ND-treated animals showed persistent dioestrus. In the androgenized groups that received MLT, ovarian morphology and size, and the number/area of corpora lutea were recovered. The number of healthy and atretic follicles was recovered when MLT was administered prior to ND; this was similar to the ovaries of control and MLT groups. There was a decrease in estrogen receptors immunostaining in the follicles of androgenized rats that were treated with MLT, and pretreatment with MLT reduced the expression of androgen receptor in atretic follicles and corpora lutea, when compared with ND-treated group. We conclude that MLT treatment recovered the histopathological aspects of the androgenized ovaries, and MLT pretreatment was the most effective.

P-MAPA immunotherapy potentiates the effect of cisplatin on serous ovarian carcinoma through targeting TLR4 signaling.

BACKGROUND: Toll-like receptors (TLRs) are transmembrane proteins expressed on the surface of ovarian cancer (OC) and immune cells. Identifying the specific roles of the TLR-mediated signaling pathways in OC cells is important to guide new treatments. Because immunotherapies have emerged as the adjuvant treatment for patients with OC, we investigated the effect of a promising immunotherapeutic strategy based on protein aggregate magnesium-ammonium phospholinoleate-palmitoleate anhydride (P-MAPA) combined with cisplatin (CIS) on the TLR2 and TLR4 signaling pathways via myeloid differentiation factor 88 (MyD88) and TLR-associated activator of interferon (TRIF) in an in vivo model of OC. METHODS: Tumors were chemically induced by a single injection of 100 µg of 7,12-dimethylbenz(a)anthracene (DMBA) directly under the left ovarian bursa in Fischer 344 rats. After the rats developed serous papillary OC, they were given P-MAPA, CIS or the combination P-MAPA+CIS as therapies. To understand the effects of the treatments, we assessed the tumor size, histopathology, and the TLR2- and TLR4-mediated inflammatory responses. RESULTS: Although CIS therapy was more effective than P-MAPA in reducing the tumor size, P-MAPA immunotherapy significantly increased the expressions of TLR2 and TLR4. More importantly, the combination of P-MAPA with CIS showed a greater survival rate compared to CIS alone, and exhibited a significant reduction in tumor volume compared to P-MAPA alone. The combination therapy also promoted the increase in the levels of the following OC-related proteins: TLR4, MyD88, TRIF, inhibitor of phosphorylated NF-kB alpha (p-IkBα), and nuclear factor kappa B (NF-kB p65) in both cytoplasmic and nuclear sites. While P-MAPA had no apparent effect on tumor necrosis factor alpha (TNF-α) and interleukin (IL)-6, it seems to increase interferon-γ (IFN-γ), which may induce the Thelper (Th1)-mediated immune response. CONCLUSION: Collectively, our results suggest that P-MAPA immunotherapy combined with cisplatin could be considered an important therapeutic strategy against OC cells based on signaling pathways activated by TLR4.

Ovarian sex steroid receptors and sex hormones in androgenized rats.

This study evaluated for the first time the effects of different doses of the anabolic steroid nandrolone decanoate (ND) on the expression of ovarian steroid receptors (AR, ER-α (ESR1) and ER-ß (ESR2)) and related sex hormones after treatment and recovery periods in adult rats. The animals were injected subcutaneously with doses of ND (1.87, 3.75, 7.5 or 15 mg/kg b.w.) or mineral oil (control group) for 15 days, and the experimental groups were divided into three periods of evaluation: (a) ND treatment for 15 days, (b) ND treatment and recovery for a period of 30 days and (c) ND treatment and recovery for a period of 60 days. Estrous cycle was monitored daily. At the end of each period, rats were killed for collection of blood and ovaries. Persistent diestrus occurred in all rats during ND treatment and after 30-day recovery. The highest dose of ND was able to maintain all rats arrested at diestrus until 60-day recovery. The expression of steroid receptors varied in a dose- and period-dependent manner, having a more pronounced response with the dose of 15 mg ND/kg. ND treatment increased serum levels of testosterone, 17ß-estradiol and dihydrotestosterone, especially at the highest doses of 7.5 and 15 mg ND/kg. No change was observed in the levels of follicle-stimulating hormone (FSH), whereas levels of the luteinizing hormone (LH) varied according to the dose and period. In conclusion, the ovarian sex steroid receptors and sex hormones were restored only at lower doses of ND and after a longer period of recovery.