Focused ultrasound on the substantia nigra enables safe neurotensin-polyplex nanoparticle-mediated gene delivery to dopaminergic neurons intranasally and by blood circulation.

Neurotensin-polyplex nanoparticles provide efficient gene transfection of nigral dopaminergic neurons when intracerebrally injected in preclinical trials of Parkinson's disease because they do not cross the blood-brain barrier (BBB). Therefore, this study aimed to open BBB with focused ultrasound (FUS) on the substantia nigra to attain systemic and intranasal transfections and evaluate its detrimental effect in rats. Systemically injected Evans Blue showed that a two-pulse FUS opened the nigral BBB. Accordingly, 35 µL of neurotensin-polyplex nanoparticles encompassing the green fluorescent protein plasmid (79.6 nm mean size and + 1.3 mV Zeta-potential) caused its expression in tyrosine hydroxylase(+) cells (dopaminergic neurons) of both substantiae nigrae upon delivery via internal carotid artery, retro-orbital venous sinus, or nasal mucosa 30 min after FUS. The intracarotid delivery yielded the highest transgene expression, followed by intranasal and venous administration. However, FUS caused neuroinflammation displayed by infiltrated lymphocytes (positive to cluster of differentiation 45), activated microglia (positive to ionized calcium-binding adaptor molecule 1), neurotoxic A1 astrocytes (positive to glial fibrillary acidic protein and complement component 3), and neurotrophic A2 astrocytes (positive to glial fibrillary acidic protein and S100 calcium-binding protein A10), that ended 15 days after FUS. Dopaminergic neurons and axonal projections decreased but recuperated basal values on day 15 after transfection, correlating with a decrease and recovery of locomotor behavior. In conclusion, FUS caused transient neuroinflammation and reversible neuronal affection but allowed systemic and intranasal transfection of dopaminergic neurons in both substantiae nigrae. Therefore, FUS could advance neurotensin-polyplex nanotechnology to clinical trials for Parkinson's disease.

Transfection of the BDNF Gene in the Surviving Dopamine Neurons in Conjunction with Continuous Administration of Pramipexole Restores Normal Motor Behavior in a Bilateral Rat Model of Parkinson's Disease.

In Parkinson's disease (PD), progressive degeneration of nigrostriatal innervation leads to atrophy and loss of dendritic spines of striatal medium spiny neurons (MSNs). The loss disrupts corticostriatal transmission, impairs motor behavior, and produces nonmotor symptoms. Nigral neurons express brain-derived neurotropic factor (BDNF) and dopamine D3 receptors, both protecting the dopamine neurons and the spines of MSNs. To restore motor and nonmotor symptoms to normality, we assessed a combined therapy in a bilateral rat Parkinson's model, with only 30% of surviving neurons. The preferential D3 agonist pramipexole (PPX) was infused for four ½ months via mini-osmotic pumps and one month after PPX initiation; the BDNF-gene was transfected into the surviving nigral cells using the nonviral transfection NTS-polyplex vector. Overexpression of the BDNF-gene associated with continuous PPX infusion restored motor coordination, balance, normal gait, and working memory. Recovery was also related to the restoration of the average number of dendritic spines of the striatal projection neurons and the number of TH-positive neurons of the substantia nigra and ventral tegmental area. These positive results could pave the way for further clinical research into this promising therapy.

Unilateral rNurr1-V5 transgene expression in nigral dopaminergic neurons mitigates bilateral neuropathology and behavioral deficits in parkinsonian rats with α-synucleinopathy.

JOURNAL/nrgr/04.03/01300535-202409000-00039/figure1/v/2024-01-16T170235Z/r/image-tiff Parkinsonism by unilateral, intranigral ß-sitosterol ß-D-glucoside administration in rats is distinguished in that the α-synuclein insult begins unilaterally but spreads bilaterally and increases in severity over time, thus replicating several clinical features of Parkinson's disease, a typical α-synucleinopathy. As Nurr1 represses α-synuclein, we evaluated whether unilateral transfected of rNurr1-V5 transgene via neurotensin-polyplex to the substantia nigra on day 30 after unilateral ß-sitosterol ß-D-glucoside lesion could affect bilateral neuropathology and sensorimotor deficits on day 30 post-transfection. This study found that rNurr1-V5 expression but not that of the green fluorescent protein (the negative control) reduced ß-sitosterol ß-D-glucoside-induced neuropathology. Accordingly, a bilateral increase in tyrosine hydroxylase-positive cells and arborization occurred in the substantia nigra and increased tyrosine hydroxylase-positive ramifications in the striatum. In addition, tyrosine hydroxylase-positive cells displayed less senescence marker ß-galactosidase and more neuron-cytoskeleton marker ßIII-tubulin and brain-derived neurotrophic factor. A significant decrease in activated microglia (positive to ionized calcium-binding adaptor molecule 1) and neurotoxic astrocytes (positive to glial fibrillary acidic protein and complement component 3) and increased neurotrophic astrocytes (positive to glial fibrillary acidic protein and S100 calcium-binding protein A10) also occurred in the substantia nigra. These effects followed the bilateral reduction in α-synuclein aggregates in the nigrostriatal system, improving sensorimotor behavior. Our results show that unilateral rNurr1-V5 transgene expression in nigral dopaminergic neurons mitigates bilateral neurodegeneration (senescence and loss of neuron-cytoskeleton and tyrosine hydroxylase-positive cells), neuroinflammation (activated microglia, neurotoxic astrocytes), α-synuclein aggregation, and sensorimotor deficits. Increased neurotrophic astrocytes and brain-derived neurotrophic factor can mediate the rNurr1-V5 effect, supporting its potential clinical use in the treatment of Parkinson's disease.

LPS Triggers Acute Neuroinflammation and Parkinsonism Involving NLRP3 Inflammasome Pathway and Mitochondrial CI Dysfunction in the Rat.

Whether neuroinflammation leads to dopaminergic nigrostriatal system neurodegeneration is controversial. We addressed this issue by inducing acute neuroinflammation in the substantia nigra (SN) with a single local administration (5 µg/2 µL saline solution) of lipopolysaccharide (LPS). Neuroinflammatory variables were assessed from 48 h to 30 days after the injury by immunostaining for activated microglia (Iba-1 +), neurotoxic A1 astrocytes (C3 + and GFAP +), and active caspase-1. We also evaluated NLRP3 activation and Il-1ß levels by western blot and mitochondrial complex I (CI) activity. Fever and sickness behavior was assessed for 24 h, and motor behavior deficits were followed up until day 30. On this day, we evaluated the cellular senescence marker ß-galactosidase (ß-Gal) in the SN and tyrosine hydroxylase (TH) in the SN and striatum. After LPS injection, Iba-1 (+), C3 (+), and S100A10 (+) cells were maximally present at 48 h and reached basal levels on day 30. NLRP3 activation occurred at 24 h and was followed by a rise of active caspase-1 (+), Il-1ß, and decreased mitochondrial CI activity until 48 h. A significant loss of nigral TH (+) cells and striatal terminals was associated with motor deficits on day 30. The remaining TH (+) cells were ß-Gal (+), suggesting senescent dopaminergic neurons. All the histopathological changes also appeared on the contralateral side. Our results show that unilaterally LPS-induced neuroinflammation can cause bilateral neurodegeneration of the nigrostriatal dopaminergic system and are relevant for understanding Parkinson's disease (PD) neuropathology.

Storm Surfing Fatalities.

OBJECTIVES: Tropical storms and hurricanes often produce ocean wave conditions that attract surfers. The risk for serious injury or death from "storm surfing" has not yet been described in the medical literature. This study aimed to quantify deaths attributable to storm surfing along the coasts of North America and the Caribbean islands. METHODS: This was a retrospective review of cases of surfing-related fatalities reported in the tropical cyclone reports of the National Hurricane Center from 1995 to 2020. Media reports were used to identify additional cases and, when available, to provide supplemental demographic and geographical information. RESULTS: There have been 27 reported storm surfing deaths during this time period. Among those for whom demographic data were available, all of the decedents were male and three were children younger than 18 years old. All but three of the deaths occurred among those surfing Atlantic storms, and one-third of the fatal injuries occurred off the coast of Florida. Eight deaths occurred while surf conditions were affected by tropical storms that did not reach hurricane status. CONCLUSIONS: Both tropical storms and hurricanes produce dangerous surf conditions that have resulted in fatal injuries among surfers during the past 25 years.

Cerebral dopamine neurotrophic factor transfection in dopamine neurons using neurotensin-polyplex nanoparticles reverses 6-hydroxydopamine-induced nigrostriatal neurodegeneration.

Overexpression of neurotrophic factors in nigral dopamine neurons is a promising approach to reverse neurodegeneration of the nigrostriatal dopamine system, a hallmark in Parkinson's disease. The human cerebral dopamine neurotrophic factor (hCDNF) has recently emerged as a strong candidate for Parkinson's disease therapy. This study shows that hCDNF expression in dopamine neurons using the neurotensin-polyplex nanoparticle system reverses 6-hydroxydopamine-induced morphological, biochemical, and behavioral alterations. Three independent electron microscopy techniques showed that the neurotensin-polyplex nanoparticles containing the hCDNF gene, ranging in size from 20 to 150 nm, enabled the expression of a secretable hCDNF in vitro. Their injection in the substantia nigra compacta on day 21 after the 6-hydroxydopamine lesion resulted in detectable hCDNF in dopamine neurons, whose levels remained constant throughout the study in the substantia nigra compacta and striatum. Compared with the lesioned group, tyrosine hydroxylase-positive (TH+) nigral cell population and TH+ fiber density rose in the substantia nigra compacta and striatum after hCDNF transfection. An increase in ßIII-tubulin and growth-associated protein 43 phospho-S41 (GAP43p) followed TH+ cell recovery, as well as dopamine and its catabolite levels. Partial reversal (80%) of drug-activated circling behavior and full recovery of spontaneous motor and non-motor behavior were achieved. Brain-derived neurotrophic factor recovery in dopamine neurons that also occurred suggests its participation in the neurotrophic effects. These findings support the potential of nanoparticle-mediated hCDNF gene delivery to develop a disease-modifying treatment against Parkinson's disease. The Institutional Animal Care and Use Committee of Centro de Investigación y de Estudios Avanzados approved our experimental procedures for animal use (authorization No. 162-15) on June 9, 2019.

Material de injerto en elevación de seno de acceso lateral: ¿es necesario? Parte II / Graft material in lateral sinus lift, is it necessary?: Part II

Introducción: Las extracciones dentarias producen una reabsorción del proceso alveolar en sentido horizontal y vertical además de la neumatización del seno. Estos cambios óseos en la zona posterior del maxilar pueden comprometer la colocación de implantes. La técnica convencional o de acceso lateral sigue siendo la técnica más utilizada para realizar elevaciones de seno maxilar. Estas elevaciones se realizan tanto sin injertos óseos como con diferentes biomateriales. Existe cierto debate sobre si es necesario colocar o no injerto. Objetivo: Analizar y comparar la ganancia ósea en elevaciones de seno de acceso lateral con injerto y sin injerto. Material y método: Se realizó una búsqueda electrónica para la actualización del tema en tres bases de datos y una serie de libros relacionados. Resultados: Se obtiene una ganancia de 4,0-6,14 mm cuando no se emplea material de injerto y entre 3,11-13,1 mm cuando se emplean biomateriales. Los estudios reflejan una pérdida ósea marginal entre 1,01-1,9 mm cuando no se emplea material de injerto y 2,3 mm de media cuando se emplean biomateriales. La técnica de elevación de seno de acceso lateral sin empleo de biomaterial presenta unas tasas de supervivencia elevadas pero las tasas de supervivencia son ligeramente superiores cuando se emplean biomateriales. La tasa de complicaciones es baja para ambas técnicas, siendo la más frecuente la perforación de la membrana de Schneider, que no parece suponer un impedimento para la colocación de implantes. Conclusión: la técnica de elevación convencional con relleno obtiene mayor ganancia ósea, pero mayor pérdida ósea marginal y presenta una tasa de supervivencia alta de implantes en comparación con la técnica que no emplea material de injerto, por lo que se debe individualizar cada caso para decidir si es necesario o no la utilización de un sustituto óseo. (AU)

Introduction: dental extractions produce a resorption of the alveolar process, horizontally and vertically in addition to pneumatization of the sinus. These bone changes in the posterior area of the maxilla can compromise implant placement. The conventional or lateral access technique is still the most used technique to perform maxillary sinus elevations. These elevations are performed without bone grafts and with different graft biomaterials where there is some discussion whether or not it is necessary to place a graft. Objective: Analyze and compare bone gain in graft and non-graft lateral access sinus lifts. Material and method: An electronic search was carried out to update the subject in three databases and a series of related books. Results: A gain of 4.0-6.14 mm is obtained when no graft material is used and between 3.11-13.1 mm when biomaterials are used. Studies show a marginal bone loss between 1.01-1.9 mm when no graft material is used and 2.3 mm on average when biomaterials are used. Lateral access sinus elevation technique without the use of biomaterial has high survival rates but survival rates are slightly higher when biomaterials are used. Rate of complications is low for both techniques, the most frequent being the perforation of Schneider’s membrane, which doesn’t seem to be an impediment to the placement of implants. Conclusion: conventional lift technique with filling obtains greater bone gain, but greater marginal bone loss and presents a high implant survival rate compared to the technique that doesn’t use graft material, so each case must be individualized to decide if it’s necessary or not the use of a bone substitute. (AU)

Regeneración ósea con injertos intraorales: rama vs. mentón. Puesta al día / Bone regeneration with intraoral graft: ramus vs. chin. Updating

La utilización de injertos intraorales en bloque es una alternativa de tratamiento válida para la regeneración en anchura de defectos óseos maxilares y mandibulares. Sin embargo, actualmente no hay consenso entre los diferentes autores en la elección del mejor tipo de bloque intraoral a utilizar. Por ende, esta puesta al día busca comparar la ganancia ósea, la tasa de complicaciones postoperatorias y el éxito del injerto entre bloques autólogos de rama mandibular y mentón. La ganancia ósea alcanzada es similar en ambos bloques. No obstante, se podría deducir una mayor ganancia al utilizar bloques de la rama mandibular cuando son evaluados mediante CBCT. Además, la tasa de reabsorción ósea fue menor con los injertos de rama. La supervivencia de los implantes es equiparable con ambos tipos de injertos. Las complicaciones que tienen lugar, en orden de frecuencia, son las alteraciones sensoriales, las necrosis pulpares, dehiscencias y hemorragias; apareciendo con mayor frecuencia en los bloques de mentón. Además, el periodo de recuperación en las zonas de rama mandibular es más lento. De este modo, a la hora de la elección parece razonable individualizar el caso y tener en consideración aspectos como la morbilidad y el acceso a la zona donante. (AU)

The use of intraoral block grafts is a valid treatment alternative for the regeneration of maxillary and mandibular horizontal bone defects. However, there is currently no consensus among different authors on the choice of the best type of intraoral bone block to use. Therefore, this update seeks to compare bone gain, post-operative complication rate and grafting success between autologous mandibular ramus and chin bone block grafts. The bone gain achieved is similar in both block grafts. However, a higher gain can be observed by CBCT when using mandibular ramus blocks. In addition, the rate of bone resorption is lower with ramus grafts. Implant survival is comparable in both types of grafts. The complications that occur, in order of frequency, are sensory alterations, pulp necrosis, dehiscence and bleeding, appearing more frequently in chin blocks. In addition, the recovery period in the mandibular ramus areas is slower. Thus, when choosing, it seems reasonable to individualize the case and take into consideration aspects such as morbidity and access to the donor area. (AU)

Virtually Possible: Medical Student Rehabilitation Rotations During a Pandemic.

ABSTRACT: The novel coronavirus 2019 pandemic has led to new dilemmas in medical education because of an initial shortage of personal protective equipment, uncertainty regarding disease transmission and treatments, travel restrictions, and social distancing guidelines. These new problems further compound the already existing problem of limited medical student exposure to the field of physical medicine and rehabilitation, particularly for students in medical schools lacking a department of physical medicine and rehabilitation, approximately 50% of medical schools. A virtual medical student physical medicine and rehabilitation rotation was created to mitigate coronavirus 2019-related limitations and impact on medical education. Using audiovisual technology, students had the opportunity to participate in clinical inpatient and outpatient care, live-streamed procedures, and virtual didactics, develop and showcase their clinical knowledge and reasoning skills, and become familiar with the culture of the physical medicine and rehabilitation residency program. Adaptive educational approaches, including integration of the flipped classroom model, success, pitfalls, and areas for improvement will be described and discussed. Providing nontraditional methods for physical medicine and rehabilitation education and exposure to medical students is crucial to maintain and promote growth of the field in this unprecedented and increasingly virtual era.

Extended Neuralgic Amyotrophy Syndrome in a Confirmed COVID-19 Patient After Intensive Care Unit and Inpatient Rehabilitation Stay.

ABSTRACT: The cause of neuralgic amyotrophy is often unknown but is commonly associated with a recent upper respiratory viral tract infection. Since the beginning of the COVID-19 pandemic, there has been a tireless effort to understand the sequelae of the virus. A 46-yr-old woman who presented after a COVID-19 hospitalization complicated by hypoxic respiratory failure requiring intubation and mechanical ventilation for 23 days was subsequently found to have lower limb sensorium changes as well as upper limb weakness. Left shoulder abduction and extension were both 3/5 in motor strength, and left hip flexion strength was 4/5 with diminished sensation to crude touch in the left lateral thigh. Nerve conduction studies and electromyography findings included a mild left median neuropathy at the wrist and motor unit recruitment pattern consistent with a chronic left upper trunk plexopathy with reinnervation. The case presented describes an extended neuralgic amyotrophy syndrome from an atraumatic mechanism in a previously diagnosed COVID-19 patient. An extended neuralgic amyotrophy syndrome has at least three immune mediated etiologies postulated (1) direct neuropathogenicity, (2) molecular mimicry, and (3) direct cytotoxic effects on peripheral nerves. As COVID-19 survivors continue to be seen in outpatient settings, practitioners should remain aware of diffuse neurological complications as sequelae of the virus persist.

Intranigral Administration of ß-Sitosterol-ß-D-Glucoside Elicits Neurotoxic A1 Astrocyte Reactivity and Chronic Neuroinflammation in the Rat Substantia Nigra.

Chronic consumption of ß-sitosterol-ß-D-glucoside (BSSG), a neurotoxin contained in cycad seeds, leads to Parkinson's disease in humans and rodents. Here, we explored whether a single intranigral administration of BSSG triggers neuroinflammation and neurotoxic A1 reactive astrocytes besides dopaminergic neurodegeneration. We injected 6 µg BSSG/1 µL DMSO or vehicle into the left substantia nigra and immunostained with antibodies against tyrosine hydroxylase (TH) together with markers of microglia (OX42), astrocytes (GFAP, S100ß, C3), and leukocytes (CD45). We also measured nitric oxide (NO), lipid peroxidation (LPX), and proinflammatory cytokines (TNF-α, IL-1ß, IL-6). The Evans blue assay was used to explore the blood-brain barrier (BBB) permeability. We found that BSSG activates NO production on days 15 and 30 and LPX on day 120. Throughout the study, high levels of TNF-α were present in BSSG-treated animals, whereas IL-1ß was induced until day 60 and IL-6 until day 30. Immunoreactivity of activated microglia (899.0 ± 80.20%) and reactive astrocytes (651.50 ± 11.28%) progressively increased until day 30 and then decreased to remain 251.2 ± 48.8% (microglia) and 91.02 ± 39.8 (astrocytes) higher over controls on day 120. C3(+) cells were also GFAP and S100ß immunoreactive, showing they were neurotoxic A1 reactive astrocytes. BBB remained permeable until day 15 when immune cell infiltration was maximum. TH immunoreactivity progressively declined, reaching 83.6 ± 1.8% reduction on day 120. Our data show that BSSG acute administration causes chronic neuroinflammation mediated by activated microglia, neurotoxic A1 reactive astrocytes, and infiltrated immune cells. The severe neuroinflammation might trigger Parkinson's disease in BSSG intoxication.

Rehabilitation of Alien Hand Syndrome Complicated by Contralateral Limb Apraxia.

A previously independent 66-yr-old right-handed man presented with right-sided weakness, preferring the lower limbs with additional impaired gait and dysarthria for 1-day duration. Imaging found a large left hemispheric anterior cerebral artery ischemic infarction with multiple lacunar infarcts. He exhibited frontal, callosal, and posterior variants of alien hand syndrome, which impeded activities of daily living. Though limited in evidence, a trial of clonazepam was initiated based on previous case reports describing suspected efficacy. Botulinum toxin A was not used given the patient's immediate need and limited hospital length of stay. Right upper limb constricting therapies improved intermanual conflict and spontaneous grasping and levitation (arm elevation in retroflexion) activity; however, concomitant left upper limb motor apraxia complicated task-oriented activities. The combination of pharmaceutical and therapeutic interventions improved the patient's quality of life as assessed by clinical observation, functional independence measures from 41 to 57, and patient reporting. This case report aims to increase awareness of a potential barrier to rehabilitation of a debilitating and rare condition and to discuss current assessment tools and treatment options supported by available evidence.

Safety Gear Decontamination Practices Among Florida Firefighters: Analysis of a Text-Based Survey Methodology.

Despite the National Fire Protection Association (NFPA) 1851 Personal Protective Equipment Care and Maintenance guidelines, little is known about the routine cleaning of firefighter bunker gear. In collaboration with a large Florida firefighter union, a mobile phone text survey was administered, which included eight questions in an item logic format. In total, 250 firefighters participated in the survey of which 65% reported cleaning their bunker gear in the past 12 months. Approximately 32% ( n = 52) indicated that they had above average confidence in gear cleaning procedures. Arriving at a fire incident response was a significant predictor of gear cleaning in the 12 months preceding survey administration. Using mobile phone-based texting for periodic queries on adherence to NFPA cleaning guidelines and safety message distribution may assist firefighters to increase decontamination procedure frequency.

Development of a Parenteral Formulation of NTS-Polyplex Nanoparticles for Clinical Purpose.

Neurotensin (NTS)-polyplex is a nanoparticle system for targeted gene delivery that holds great promise for treatment of Parkinson's disease and various types of cancer. However, the high instability in aqueous suspension of NTS-polyplex nanoparticles is a major limitation for their widespread clinical use. To overcome this obstacle, we developed a clinical formulation and a lyophilization process for NTS-polyplex nanoparticles. The reconstituted samples were compared with fresh preparations by using transmission electron microscopy, dynamic light scattering, electrophoretic mobility, circular dichroism and transfection assays in vitro and in vivo. Our formulation was able to confer lyoprotection and stability to these nanoparticles. In addition, transmission electron microscopy (TEM) and size exclusion-high performance liquid chromatography (SEC-HPLC) using a radioactive tag revealed that the interaction of reconstituted nanoparticles with fetal bovine or human serum did not alter their biophysical features. Furthermore, the formulation and the lyophilization procedure guaranteed functional NTS-polyplex nanoparticles for at least six months of storage at 25 °C and 60% relative humidity. Our results offer a pharmaceutical guide for formulation and long-term storage of NTS-polyplex nanoparticles that could be applied to other polyplexes.

Neurturin overexpression in dopaminergic neurons induces presynaptic and postsynaptic structural changes in rats with chronic 6-hydroxydopamine lesion.

The structural effect of neurturin (NRTN) on the nigrostriatal dopaminergic system in animals remains unknown, although NRTN has been shown to be effective in Parkinson's disease animal models. Herein, we aimed to demonstrate that NRTN overexpression in dopaminergic neurons stimulates both neurite outgrowths in the nigrostriatal pathway and striatal dendritic spines in aging rats with chronic 6-hydroxydopamine (6-OHDA) lesion. At week 12 after lesion, pTracer-mNRTN-His or pGreenLantern-1 plasmids were intranigrally transfected using the NTS-polyplex nanoparticles system. We showed that the transgenic expression in dopaminergic neurons remained until the end of the study (12 weeks). Only animals expressing NRTN-His showed recovery of tyrosine hydroxylase (TH)+ cells (28 ± 2%), their neurites (32 ± 2%) and the neuron-specific cytoskeletal marker ß-III-tubulin in the substantia nigra; striatal TH(+) fibers were also recovered (52 ± 3%), when compared to the healthy condition. Neurotensin receptor type 1 levels were also significantly recovered in the substantia nigra and striatum. Dopamine recovery was 70 ± 4% in the striatum and complete in the substantia nigra. The number of dendritic spines of striatal medium spiny neurons was also significantly increased, but the recovery was not complete. Drug-activated circling behavior decreased by 73 ± 2% (methamphetamine) and 89 ± 1% (apomorphine). Similar decrease was observed in the spontaneous motor behavior. Our results demonstrate that NRTN causes presynaptic and postsynaptic restoration of the nigrostriatal dopaminergic system after a 6-OHDA-induced chronic lesion. However, those improvements did not reach the healthy condition, suggesting that NRTN exerts lesser neurotrophic effects than other neurotrophic approaches.

Regulation of human GDNF gene expression in nigral dopaminergic neurons using a new doxycycline-regulated NTS-polyplex nanoparticle system.

The human glial-cell derived neurotrophic factor (hGDNF) gene transfer by neurotensin (NTS)-polyplex nanoparticles functionally restores the dopamine nigrostriatal system in experimental Parkinson's disease models. However, high levels of sustained expression of GDNF eventually can cause harmful effects. Herein, we report an improved NTS-polyplex nanoparticle system that enables regulation of hGDNF expression within dopaminergic neurons. We constructed NTS-polyplex nanoparticles containing a single bifunctional plasmid that codes for the reverse tetracycline-controlled transactivator advanced (rtTA-Adv) under the control of NBRE3x promoter, and for hGDNF under the control of tetracycline-response element (TRE). Another bifunctional plasmid contained the enhanced green fluorescent protein (GFP) gene. Transient transfection experiments in N1E-115-Nurr1 cells showed that doxycycline (100 ng/mL) activates hGDNF and GFP expression. Doxycycline (5 mg/kg, i.p.) administration in rats activated hGDNF expression only in transfected dopaminergic neurons, whereas doxycycline withdrawal silenced transgene expression. Our results offer a specific doxycycline-regulated system suitable for nanomedicine-based treatment of Parkinson's disease.

Neurotensin-polyplex-mediated brain-derived neurotrophic factor gene delivery into nigral dopamine neurons prevents nigrostriatal degeneration in a rat model of early Parkinson's disease.

BACKGROUND: The neurotrophin Brain-Derived Neurotrophic Factor (BDNF) influences nigral dopaminergic neurons via autocrine and paracrine mechanisms. The reduction of BDNF expression in Parkinson's disease substantia nigra (SN) might contribute to the death of dopaminergic neurons because inhibiting BDNF expression in the SN causes parkinsonism in the rat. This study aimed to demonstrate that increasing BDNF expression in dopaminergic neurons of rats with one week of 6-hydroxydopamine lesion recovers from parkinsonism. The plasmids phDAT-BDNF-flag and phDAT-EGFP, coding for enhanced green fluorescent protein, were transfected using neurotensin (NTS)-polyplex, which enables delivery of genes into the dopaminergic neurons via neurotensin-receptor type 1 (NTSR1) internalization. RESULTS: Two weeks after transfections, RT-PCR and immunofluorescence techniques showed that the residual dopaminergic neurons retain NTSR1 expression and susceptibility to be transfected by the NTS-polyplex. phDAT-BDNF-flag transfection did not increase dopaminergic neurons, but caused 7-fold increase in dopamine fibers within the SN and 5-fold increase in innervation and dopamine levels in the striatum. These neurotrophic effects were accompanied by a significant improvement in motor behavior. CONCLUSIONS: NTS-polyplex-mediated BDNF overexpression in dopaminergic neurons has proven to be effective to remit hemiparkinsonism in the rat. This BDNF gene therapy might be helpful in the early stage of Parkinson's disease.

The transfection of BDNF to dopamine neurons potentiates the effect of dopamine D3 receptor agonist recovering the striatal innervation, dendritic spines and motor behavior in an aged rat model of Parkinson's disease.

The progressive degeneration of the dopamine neurons of the pars compacta of substantia nigra and the consequent loss of the dopamine innervation of the striatum leads to the impairment of motor behavior in Parkinson's disease. Accordingly, an efficient therapy of the disease should protect and regenerate the dopamine neurons of the substantia nigra and the dopamine innervation of the striatum. Nigral neurons express Brain Derived Neurotropic Factor (BDNF) and dopamine D3 receptors, both of which protect the dopamine neurons. The chronic activation of dopamine D3 receptors by their agonists, in addition, restores, in part, the dopamine innervation of the striatum. Here we explored whether the over-expression of BDNF by dopamine neurons potentiates the effect of the activation of D3 receptors restoring nigrostriatal innervation. Twelve-month old Wistar rats were unilaterally injected with 6-hydroxydopamine into the striatum. Five months later, rats were treated with the D3 agonist 7-hydroxy-N,N-di-n-propy1-2-aminotetralin (7-OH-DPAT) administered i.p. during 4½ months via osmotic pumps and the BDNF gene transfection into nigral cells using the neurotensin-polyplex nanovector (a non-viral transfection) that selectively transfect the dopamine neurons via the high-affinity neurotensin receptor expressed by these neurons. Two months after the withdrawal of 7-OH-DPAT when rats were aged (24 months old), immunohistochemistry assays were made. The over-expression of BDNF in rats receiving the D3 agonist normalized gait and motor coordination; in addition, it eliminated the muscle rigidity produced by the loss of dopamine. The recovery of motor behavior was associated with the recovery of the nigral neurons, the dopamine innervation of the striatum and of the number of dendritic spines of the striatal neurons. Thus, the over-expression of BDNF in dopamine neurons associated with the chronic activation of the D3 receptors appears to be a promising strategy for restoring dopamine neurons in Parkinson's disease.