Amar Jyoti

Dendrimer-based postnatal therapy for neuroinflammation and cerebral palsy in a rabbit model.

A dendrimer-drug conjugate attenuates neuroinflammation and improves motor function in a rabbit model of cerebral palsy. One Hop at a Time Cerebral palsy (CP) is a developmental disorder caused by injury to a baby’s brain while it is still developing, either in the womb or during the early months of life, but is often not diagnosed until children are 2 to 3 years of age. There is no cure for CP, and the best option for affected children is intensive physical therapy to improve motor skills. Now, Kannan et al. have designed a dendrimer-based therapeutic for treating this developmental disorder in baby rabbits (kits), opening the door to new treatment options in humans. The authors chose to use the rabbit model of CP, which replicates the neuroinflammation seen in human brains as well as the motor deficits in children. To generate this model, Kannan and colleagues injected Escherichia coli toxin into the rabbit mother’s uterus at about 90% term gestation. When the kits were born, they were administered either a saline solution, a free drug known as NAC (N-acetyl-l-cysteine), or a dendrimer-NAC (D-NAC) conjugate. This postnatal “rescue” with D-NAC, given on day 1 of life, allowed CP kits to develop normally, able to walk and hop. The successfully treated kits also had neuron counts and low inflammation similar to healthy control animals. By comparison, NAC alone or saline had no effect. The authors believe that conjugating NAC to the dendrimers promoted greater uptake by activated microglia and astrocytes, with no toxicity to surrounding neurons. Although still in preclinical testing in rabbits, this dendrimer-drug conjugate shows promise for postnatal treatment of babies suspected of having CP. Cerebral palsy (CP) is a chronic childhood disorder with no effective cure. Neuroinflammation, caused by activated microglia and astrocytes, plays a key role in the pathogenesis of CP and disorders such as Alzheimer’s disease and multiple sclerosis. Targeting neuroinflammation can be a potent therapeutic strategy. However, delivering drugs across the blood-brain barrier to the target cells for treating diffuse brain injury is a major challenge. We show that systemically administered polyamidoamine dendrimers localize in activated microglia and astrocytes in the brain of newborn rabbits with CP, but not healthy controls. We further demonstrate that dendrimer-based N-acetyl-l-cysteine (NAC) therapy for brain injury suppresses neuroinflammation and leads to a marked improvement in motor function in the CP kits. The well-known and safe clinical profile for NAC, when combined with dendrimer-based targeting, provides opportunities for clinical translation in the treatment of neuroinflammatory disorders in humans. The effectiveness of the dendrimer-NAC treatment, administered in the postnatal period for a prenatal insult, suggests a window of opportunity for treatment of CP in humans after birth.

Aluminium-induced electrophysiological, biochemical and cognitive modifications in the hippocampus of aging rats

Aluminium (Al) is the most abundant metal known for its neurotoxicity in humans. It gains easy access to the central nervous system under normal physiological conditions and accumulates in different brain regions. It has been reported to be involved in the etiology of several neurodegenerative diseases. In this study, we have investigated the effects of long-term intake of aluminium chloride (AlCl(3)) on the electrophysiological, behavioral, biochemical and histochemical functions of hippocampus. Wistar rats were fed with AlCl(3) at a dose of 50mg/(kgday) for 6 months in the drinking water. Effect of long-term intake of Al was studied on the electrical activity of hippocampal CA1 and CA3 regions in brain of young and old rats. Morris water maze and open field tests were performed to investigate the cognitive and anxiety status of aging rats intoxicated with aluminium. Our studies indicate that aluminium intake results in increased multiple unit activity and adversely affect the spatial learning and memory abilities of both young and old rats. Aluminium intake also inflicts oxidative stress-related damage to lipids, membrane associated proteins (Na-K ATPase and PKC) and endogenous antioxidant enzyme activity (SOD, GPx and GST). The compromised antioxidant system might be playing a crucial role in the observed Al-induced alterations. We have observed that the magnitude of AlCl(3)-induced alteration was considerably higher in younger group of rats compared to older group. In conclusion, the results of the present study implicates that aluminium treatment exerts its neurotoxic effects by altering the overall physiology of brain, and the induced changes were strongly correlated with each other.

Curcumin protects against electrobehavioral progression of seizures in the iron-induced experimental model of epileptogenesis

The purpose of the study was to investigate whether dietary intake of curcumin can inhibit the onset and progression of seizures and their associated pathophysiology in experimental FeCl(3)-induced epileptogenesis. Curcumin was considered for this study because it can cross the blood-brain barrier and bind redox-active metal ions. It is also well known for its antioxidative, anticancer, and anti-inflammatory properties. In the present study, seizures were induced by intracortical injection of FeCl(3) into young rats. Synchronized video/EEG recordings were obtained to diagnose the progression of seizures. Short-term treatment with a curcumin-supplemented diet (1500 pp mw/w) significantly inhibited the onset of grade III and IV seizures in rats with iron-induced epilepsy. The lower dose of curcumin (500 ppm) was not effective in inhibiting grade III seizures, but retarded the onset and progression of generalized seizures. The seizure-suppressing potential of curcumin is explained by the observed biochemical, behavioral, and ultrastructural results. Our results indicate that curcumin significantly prevents generalization of electroclinical seizure activity as well as the pathogenesis associated with iron-induced epileptogenesis.

EEG, activity, and sleep architecture in a transgenic AβPPswe/PSEN1A246E Alzheimer's disease mouse.

Since sleep and electroencephalogram (EEG) disturbances are endophenotypes of Alzheimers disease (AD) patients alongside cognitive dysfunction, we here characterized these parameters in transgenic mice carrying transgenes for amyloid-β protein precursor (AβPPswe) and presenilin 1 (PSEN1A246E) at 5 (pre-plaque) and 20 months, relative to PSEN1 and wild-type (WT) mice, using a novel wireless microchip device. While circadian rhythms were not affected, we obtained significantly higher overall activity at 5 months in the AβPP/PSEN1 strain (p < 0.001) compared to both PSEN1 and WT animals. Vigilance staging revealed that AβPP/PSEN1 animals present with an age-independent increase in wakefulness (p < 0.001) and a decrease in non rapid-eye movement (NREM) sleep (p < 0.01). These changes were age- and genotype-dependent only during the light phase, while dark phase activity pattern were equally affected at both ages. In all genotypes, the amount of REM sleep was lower at 20 months indicating a general age-related profile. Spectral power of qEEG changed in AβPP/PSEN1 mice at 5 months during wakefulness and REM sleep; during wakefulness hippocampal delta (0.5-5 Hz) was reduced and theta (5-9 Hz) power enhanced. By contrast, NREM EEG spectra were affected by age and genotype. Interestingly, PSEN1 animals also showed spectral EEG changes, these differed from both WT and AβPP/PSEN1 animals. Our results indicate that AβPP/PSEN1 mice exhibit abnormalities in activity and sleep architecture preceding amyloid plaque deposition as well as age-related changes in cortical EEG power. Though not fully recapitulating the profile of AD patients, this suggests activity and EEG recordings as sensitive and translational biomarkers in murine models.

Biodistribution of Fluorescently Labeled PAMAM Dendrimers in Neonatal Rabbits: Effect of Neuroinflammation

Dendrimers are being explored in many preclinical studies as drug, gene, and imaging agent delivery systems. Understanding their detailed organ, tissue, cellular uptake, and retention can provide valuable insights into their effectiveness as delivery vehicles and the associated toxicity. This work explores a fluorescence-quantification based assay that enables simultaneous quantitative biodistribution and imaging of dendrimers with a single agent. We have labeled an ethylenediamine-core generation-4 hydroxyl-terminated poly(amidoamine) (PAMAM) dendrimer using the fluorescent photostable, near-IR cyanine dye (Cy5) and performed quantitative and qualitative biodistribution of the dendrimer-Cy5 conjugates (D-Cy5) in healthy neonatal rabbits and neonatal rabbits with cerebral palsy (CP). The biodistribution of D-Cy5 and free Cy5 dye was evaluated in newborn rabbits, based on the developed quantification methods using fluorescence spectroscopy, high-performance liquid chromatography (HPLC), and size exclusion chromatography (SEC) and supported by microscopic imaging. The uptake was assessed in the brain, heart, liver, lungs, kidneys, blood serum, and urine. Results obtained based on these three independent methods are in good agreement and indicate the fast renal clearance of D-Cy5 and free Cy5 with relatively higher organs accumulation of the D-Cy5 conjugate. Following systemic administration, the D-Cy5 mainly accumulated in kidneys and bladder at 24 h. The quantitative biodistribution is in good agreement with previous studies based on radiolabeling. These methods for dendrimers quantification are easier and more practical, provide excellent sensitivity (reaching 0.1 ng per gram of tissue), and allow for quantification of dendrimers in different organs over longer time periods without concerns for radioactive decay, while also enabling tissue and cellular imaging in the same animal. In kits with fetal-neuroinflammation induced CP, there was a significantly higher uptake of D-Cy5 in the brain, while biodistribution in other organs was similar to that of healthy kits.

Curcumin attenuates aluminium-induced functional neurotoxicity in rats

Curcumin is a polyphenol extracted from the rhizome of Curcuma longa and well known as a multi-functional drug with antioxidative, anti-cancerous and anti-inflammatory activities. Curcumins antiaging and neuroprotective potential is widely reported. In the present study, effect of curcumin treatment dose 30 mg kg(-1) day(-1) was investigated against aluminium neurotoxicity in young and old animals. Direct and indirect intakes of aluminium have been reported to be involved in the etiology of several neurodegenerative disorders like Alzheimers and Parkinsons diseases. Long term Al was administered through drinking water at a dose of 50 mg/kg/day for 6 months in both young (4 months) and old (18 months) male Wistar rats. Result obtained demonstrates that curcumin treatment attenuates the Al-induced alterations at biochemical, behavioral and ultrastructural levels which was well reflected in the electrophysiological recordings. Our results indicate that curcumins ability to bind redox active metals and cross the blood-brain barrier could be playing crucial role in preventing against Al-induced neurotoxicity.

Concurrent quantification of tryptophan and its major metabolites

An imbalance in tryptophan (TRP) metabolites is associated with several neurological and inflammatory disorders. Therefore, analytical methods allowing for simultaneous quantification of TRP and its major metabolites would be highly desirable, and may be valuable as potential biomarkers. We have developed a HPLC method for concurrent quantitative determination of tryptophan, serotonin, 5-hydroxyindoleacetic acid, kynurenine, and kynurenic acid in tissue and fluids. The method utilizes the intrinsic spectroscopic properties of TRP and its metabolites that enable UV absorbance and fluorescence detection by HPLC, without additional labeling. The origin of the peaks related to analytes of interest was confirmed by UV-Vis spectral patterns using a PDA detector and mass spectrometry. The developed methods were validated in rabbit fetal brain and amniotic fluid at gestational day 29. Results are in excellent agreement with those reported in the literature for the same regions. This method allows for rapid quantification of tryptophan and four of its major metabolites concurrently. A change in the relative ratios of these metabolites can provide important insights in predicting the presence and progression of neuroinflammation in disorders such as cerebral palsy, autism, multiple sclerosis, Alzheimer disease, and schizophrenia.

Investigating the Radioresistant Properties of Lung Cancer Stem Cells in the Context of the Tumor Microenvironment

Lung cancer is the most common cause of cancer-related deaths worldwide and non-small cell lung cancer (NSCLC) accounts for ~85% of all lung cancer. While recent research has shown that cancer stem cells (CSC) exhibit radioresistant and chemoresistant properties, current cancer therapy targets the bulk of the tumor burden without accounting for the CSC and the contribution of the tumor microenvironment. CSC interaction with the stroma enhances NSCLC survival, thus limiting the efficacy of treatment. The aim of this study was to elucidate the role of CSC and the microenvironment in conferring radio- or chemoresistance in an in vitro tumor model for NSCLC. The novel in vitro three-dimensional (3D) NSCLC model of color-coded tumor tissue analogs (TTA) that we have developed is comprised of human lung adenocarcinoma cells, fibroblasts, endothelial cells and NSCLC cancer stem cells maintained in low oxygen conditions (5% O2) to recapitulate the physiologic conditions in tumors. Using this model, we demonstrate that a single 5 Gy radiation dose does not inhibit growth of TTA containing CSC and results in elevated expression of cytokines (TGF-α, RANTES, ENA-78) and factors (vimentin, MMP and TIMP), indicative of an invasive and aggressive phenotype. However, combined treatment of single dose or fractionated doses with cisplatin was found to either attenuate or decrease the proliferative effect that radiation exposure alone had on TTA containing CSC maintained in hypoxic conditions. In summary, we utilized a 3D NSCLC model, which had characteristics of the tumor microenvironment and tumor cell heterogeneity, to elucidate the multifactorial nature of radioresistance in tumors.

Aging accelerates the progression and manifestation of seizures in post-traumatic model of epilepsy.

Traumatic brain injury is a major risk of post-traumatic epilepsy in a large number of individuals of different age groups. Lots of research has been done to elucidate the mechanism of post-traumatic epileptogenesis but age-related vulnerability to develop traumatic seizures is still unknown. Therefore, in the present study investigations were carried out to characterize the electrobehavioral seizure manifestation and associated alterations in young and old epileptic groups. FeCl(3) injection model was used to induce post-traumatic seizures as this model closely resembles human post-traumatic epilepsy. Synchronized video-EEG monitoring was performed to diagnose manifestation of seizures in young (4 months) and old (18 months) rats. Biochemical and ultrastructural studies were performed to determine the mechanism behind the altered age-related vulnerability for post-traumatic seizures. Our result shows that old rats were more vulnerable to post-traumatic epilepsy due to faster seizure spread and lower latency for generalization of electro-clinical seizure activity. The observed biochemical and microscopic alterations associated with old age positively correlate with the altered susceptibility to develop seizures in old epileptic groups.

An in vitro assessment of liposomal topotecan simulating metronomic chemotherapy in combination with radiation in tumor-endothelial spheroids.

Low dose metronomic chemotherapy (LDMC) refers to prolonged administration of low dose chemotherapy designed to minimize toxicity and target the tumor endothelium, causing tumor growth inhibition. Topotecan (TPT) when administered at its maximum tolerated dose (MTD) is often associated with systemic hematological toxicities. Liposomal encapsulation of TPT enhances efficacy by shielding it from systemic clearance, allowing greater uptake and extended tissue exposure in tumors. Extended release of TPT from liposomal formulations also has the potential to mimic metronomic therapies with fewer treatments. Here we investigate potential toxicities of equivalent doses of free and actively loaded liposomal TPT (LTPT) and compare them to a fractionated low dose regimen of free TPT in tumor-endothelial spheroids (TES) with/without radiation exposure for a prolonged period of 10 days. Using confocal microscopy, TPT fluorescence was monitored to determine the accumulation of drug within TES. These studies showed TES, being more reflective of the in vivo tumor microenvironment, were more sensitive to LTPT in comparison to free TPT with radiation. More importantly, the response of TES to low-dose metronomic TPT with radiation was comparable to similar treatment with LTPT. This TES study suggests nanoparticle formulations designed for extended release of drug can simulate LDMC in vivo.