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1.
Nat Methods ; 21(1): 132-141, 2024 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-38129618

RESUMEN

Multiphoton microscopy can resolve fluorescent structures and dynamics deep in scattering tissue and has transformed neural imaging, but applying this technique in vivo can be limited by the mechanical and optical constraints of conventional objectives. Short working distance objectives can collide with compact surgical windows or other instrumentation and preclude imaging. Here we present an ultra-long working distance (20 mm) air objective called the Cousa objective. It is optimized for performance across multiphoton imaging wavelengths, offers a more than 4 mm2 field of view with submicrometer lateral resolution and is compatible with commonly used multiphoton imaging systems. A novel mechanical design, wider than typical microscope objectives, enabled this combination of specifications. We share the full optical prescription, and report performance including in vivo two-photon and three-photon imaging in an array of species and preparations, including nonhuman primates. The Cousa objective can enable a range of experiments in neuroscience and beyond.


Asunto(s)
Colorantes , Microscopía de Fluorescencia por Excitación Multifotónica , Animales , Microscopía de Fluorescencia por Excitación Multifotónica/métodos
2.
Drug Deliv ; 31(1): 2379369, 2024 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-39010743

RESUMEN

PURPOSE: Targeted drug delivery to the optic nerve head may be useful in the preclinical study and later clinical management of optic neuropathies, however, there are no FDA-approved drug delivery systems to achieve this. The purpose of this work was to develop an optic nerve head drug delivery technique. METHODS: Different strategies to approach the optic nerve head were investigated, including standard intravitreal and retroorbital injections. A novel SupraChoroidal-to-Optic-NervE (SCONE) delivery was optimized by creating a sclerotomy and introducing a catheter into the suprachoroidal space. Under direct visualization, the catheter was guided to the optic nerve head. India ink was injected. The suprachoroidal approach was performed in New Zealand White rabbit eyes in vivo (25 animals total). Parameters, including microneedle size and design, catheter design, and catheter tip angle, were optimized ex vivo and in vivo. RESULTS: Out of the candidate optic nerve head approaches, intravitreal, retroorbital, and suprachoroidal approaches were able to localize India ink to within 2 mm of the optic nerve. The suprachoroidal approach was further investigated, and after optimization, was able to deposit India ink directly within the optic nerve head in up to 80% of attempts. In eyes with successful SCONE delivery, latency and amplitude of visual evoked potentials was not different than the naïve untreated eye. CONCLUSIONS: SCONE delivery can be used for targeted drug delivery to the optic nerve head of rabbits without measurable toxicity measured anatomically or functionally. Successful development of this system may yield novel opportunities to study optic nerve head-specific drug delivery in animal models, and paradigm-shifting management strategies for treating optic neuropathies. TRANSLATIONAL RELEVANCE: Here we demonstrate data on a new method for targeted delivery to the optic nerve head, addressing a significant unmet need in therapeutics for optic neuropathies.


Asunto(s)
Sistemas de Liberación de Medicamentos , Animales , Conejos , Coroides , Nervio Óptico/efectos de los fármacos , Potenciales Evocados Visuales/efectos de los fármacos , Disco Óptico , Inyecciones Intravítreas , Agujas , Carbono
3.
JCI Insight ; 9(3)2024 Feb 08.
Artículo en Inglés | MEDLINE | ID: mdl-38194296

RESUMEN

Traumatic optic neuropathy (TON) is a devastating condition that can occur after blunt or penetrating trauma to the head, leading to visual impairment or blindness. Despite these debilitating effects, no clinically available therapeutic targets neuroprotection or promotes axon regeneration in this or any optic neuropathy. Limited data in large-animal models are a major obstacle to advancing treatments toward clinical therapeutics. To address this issue, we refined a surgical model of TON in Yucatan minipigs. First, we validated the model by demonstrating visual impairment by flash visual-evoked potential and retinal ganglion cell degeneration and death. Next, we developed and optimized a delivery method and nontoxic dosing of intravitreal brain-derived neurotrophic factor (BDNF) and cAMP. Finally, we showed that intravitreal injection of BDNF and cAMP rescued visual function and protected against retinal ganglion cell death and optic nerve axon degeneration. Together these data in a preclinical large-animal model advance our understanding of and ability to model TON and further identify and develop candidate clinical therapeutics.


Asunto(s)
Factor Neurotrófico Derivado del Encéfalo , Traumatismos del Nervio Óptico , Animales , Porcinos , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Traumatismos del Nervio Óptico/tratamiento farmacológico , Axones/metabolismo , Neuroprotección , Regeneración Nerviosa , Porcinos Enanos/metabolismo , Trastornos de la Visión
4.
Epilepsia ; 54(9): 1535-41, 2013 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-23848506

RESUMEN

PURPOSE: The role of granule cell axon (mossy fiber) sprouting in temporal lobe epileptogenesis is unclear and controversial. Rapamycin suppresses mossy fiber sprouting, but its reported effects on seizure frequency are mixed. The present study used high-dose rapamycin to more completely block mossy fiber sprouting and to measure the effect on seizure frequency. METHODS: Mice were treated with pilocarpine to induce status epilepticus. Beginning 24 h later and continuing for 2 months, vehicle or rapamycin (10 mg/kg/day) was administered. Starting 1 month after status epilepticus, mice were monitored by video 9 h per day, every day, for 1 month to measure the frequency of spontaneous motor seizures. At the end of seizure monitoring, a subset of mice was prepared for anatomic analysis. Mossy fiber sprouting was measured as the proportion of the granule cell layer and molecular layer that displayed black labeling in Timm-stained sections. KEY FINDINGS: Extensive mossy fiber sprouting developed in mice that experienced status epilepticus and were treated with vehicle. In rapamycin-treated mice, mossy fiber sprouting was blocked almost to the level of naive controls. Seizure frequency was similar in vehicle-treated and rapamycin-treated mice. SIGNIFICANCE: These findings suggest that mossy fiber sprouting is not necessary for epileptogenesis in the mouse pilocarpine model. They also reveal that rapamycin does not have antiseizure or antiepileptogenic effects in this model.


Asunto(s)
Epilepsia del Lóbulo Temporal/tratamiento farmacológico , Fibras Musgosas del Hipocampo/efectos de los fármacos , Neuronas/efectos de los fármacos , Sirolimus/uso terapéutico , Animales , Axones/efectos de los fármacos , Modelos Animales de Enfermedad , Femenino , Masculino , Ratones , Pilocarpina/administración & dosificación , Sirolimus/administración & dosificación
5.
Dev Dyn ; 241(2): 365-75, 2012 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-22223620

RESUMEN

BACKGROUND: Tbx2, Tbx3, Tbx4, and Tbx5, members of the Tbx2 subfamily of T-box transcription factor genes, are important for many aspects of embryonic development and mutations in some human TBX2 subfamily genes cause developmental syndromes. In addition, TBX2 and TBX3 are overexpressed in a variety of cancers, including reproductive system cancers. This study characterizes the expression of Tbx2 subfamily genes during development of the reproductive system. RESULTS: We show that these genes are expressed in both the internal and external reproductive systems. Tbx2 is expressed in gonads and genital ducts, the Wolffian and Müllerian ducts, while Tbx3 is only expressed in genital ducts. Tbx4 is expressed in embryonic and postnatal germ cells. All four genes are expressed in mesenchyme in external genitalia, with Tbx3 and Tbx5 expression in the epithelium as well. CONCLUSION: This study lays the foundation for investigation of functional requirements for Tbx2 subfamily genes in development of the mammalian reproductive system.


Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Genitales/embriología , Diferenciación Sexual/genética , Proteínas de Dominio T Box/genética , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos ICR , Reproducción/genética
6.
J Am Assoc Lab Anim Sci ; 59(2): 170-175, 2020 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-32059754

RESUMEN

Alfaxalone is an injectable anesthetic agent that is used in veterinary medicine for general anesthesia. We evaluated the safety and efficacy of alfaxalone delivered through continuous rate infusion by comparing ketamine-xylazine-alfaxalone (KXA) anesthesia with ketamine-xylazine (KX) anesthesia in Sprague-Dawley rats. Anesthesia was induced in male and female rats by using subcutaneous KX. After induction, rats in the KXA group received alfaxalone (10 mg/kg/h IV) for 35 min, whereas rats in the KX group did not receive alfaxalone. At the end of the trial, alfaxalone was discontinued, and xylazine was reversed in all rats by using atipamezole. Throughout anesthesia, we assessed forepaw withdrawal reflex (FPWR), hindpaw withdrawal reflex (HPWR), response to surgical stimulation, heart rate, respiratory rate, SpO2, body temperature, and time to standing. KXA produced a reliable surgical plane of anesthesia, as evidenced by the loss of both FPWR and HPWR and lack of response to surgical stimulation in all 16 rats, whereas only 6 of the 16 rats in the KX group lost HPWR. No rat in the KXA group regained a paw withdrawal reflex during alfaxalone administration, whereas 3 of the 12 rats (25%) in the KX group that reached a surgical plane of anesthesia exited that plane within the 35-min timeframe. Neither heart rate, respiratory rate, SpO2, body temperature, nor time to standing differed between KXA and KX groups; and there were no sex-associated differences in anesthesia response. These results indicate that alfaxalone (10 mg/kg/h IV) delivered through continuous rate infusion, in combination with ketamine and xylazine, provides a safe, prolonged, and reliable surgical plane of anesthesia in rats.


Asunto(s)
Anestésicos/farmacología , Ketamina/farmacología , Pregnanodionas/farmacología , Xilazina/farmacología , Anestesia General/veterinaria , Anestésicos/administración & dosificación , Animales , Temperatura Corporal/efectos de los fármacos , Quimioterapia Combinada , Femenino , Frecuencia Cardíaca/efectos de los fármacos , Hipnóticos y Sedantes/administración & dosificación , Hipnóticos y Sedantes/farmacología , Ketamina/administración & dosificación , Ciencia de los Animales de Laboratorio , Masculino , Pregnanodionas/administración & dosificación , Ratas , Ratas Sprague-Dawley , Frecuencia Respiratoria/efectos de los fármacos , Xilazina/administración & dosificación
7.
Invest Ophthalmol Vis Sci ; 60(6): 1953-1966, 2019 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-31060051

RESUMEN

Purpose: Increased endoplasmic reticulum (ER) stress is one of the earliest subcellular changes in neuro-ophthalmic diseases. In this study, we investigated the expression of key molecules in the ER stress pathways following nonarteritic anterior ischemic optic neuropathy (AION), the most common acute optic neuropathy in adults over 50, and assessed the impact of chemical chaperon 4-phenylbutyric acid (4-PBA) in vivo. Methods: We induced AION using photochemical thrombosis in adult mice and performed histologic analyses of key molecules in the ER stress pathway in the retina and optic nerve. We also assessed the effects of daily intraperitoneal injections of 4-PBA after AION. Results: In the retina at baseline, there was low proapoptotic transcriptional regulator C/EBP homologous protein (CHOP) and high prosurvival chaperon glucose-regulated protein 78 (GRP78) expression in retinal ganglion cells (RGCs). One day after AION, there was significantly increased CHOP and reduced GRP78 expressions in the ganglion cell layer. In the optic nerve at baseline, there was little CHOP and high GRP78 expression. One day after AION, there was significantly increased CHOP and no change in GRP78 expression. Treatment immediately after AION using daily intraperitoneal injection of chemical chaperone 4-PBA for 19 days significantly rescued Brn3A+ RGCs and Olig2+ optic nerve oligodendrocytes. Conclusions: We showed for the first time that acute AION resulted in increased ER stress and differential expression of ER stress markers CHOP and GRP78 in the retina and optic nerve. Rescue of RGCs and oligodendrocytes with 4-PBA provides support for ER stress reduction as possible treatment for AION.


Asunto(s)
Estrés del Retículo Endoplásmico , Oligodendroglía/patología , Disco Óptico/patología , Neuropatía Óptica Isquémica/patología , Fenilbutiratos/farmacología , Células Ganglionares de la Retina/patología , Animales , Modelos Animales de Enfermedad , Chaperón BiP del Retículo Endoplásmico , Femenino , Regulación de la Expresión Génica , Proteínas de Choque Térmico/biosíntesis , Proteínas de Choque Térmico/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Chaperonas Moleculares , Oligodendroglía/metabolismo , Disco Óptico/metabolismo , Neuropatía Óptica Isquémica/tratamiento farmacológico , Neuropatía Óptica Isquémica/genética , Células Ganglionares de la Retina/metabolismo , Factor de Transcripción CHOP/biosíntesis , Factor de Transcripción CHOP/genética
8.
J Am Assoc Lab Anim Sci ; 53(6): 684-91, 2014 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-25650976

RESUMEN

Extending a surgical plane of anesthesia in mice by using injectable anesthetics typically is accomplished by repeat-bolus dosing. We compared the safety and efficacy of redosing protocols administered either during an anesthetic surgical plane (maintaining a continuous surgical plane, CSP), or immediately after leaving this plane (interrupted surgical plane, ISP) in C57BL/6J mice. Anesthesia was induced with ketamine, xylazine, and acepromazine (80, 8, and 1 mg/kg IP, respectively), and redosing protocols included 25% (0.25K), 50% (0.5K), or 100% (1.0K) of the initial ketamine dose or 25% (0.25KX) or 50% (0.5KX) of the initial ketamine-xylazine dose. In the ISP group, the surgical plane was extended by 13.8 ± 2.1 min (mean ± SEM) after redosing for the 0.25K redose with 50% returning to a surgical plane, 42.7 ± 4.5 min for the 0.5K redose with 88% returning to a surgical plane, and 44.3 ± 15.4 min for the 1.0K redose, 52.8 ± 7.2 min for the 0.25KX redose, and 45.9 ± 2.9 min for the 0.5KX redose, with 100% of mice returning to a surgical plane of anesthesia in these 3 groups. Mortality rates for ISP groups were 0%, 12%, 33%, 12%, and 18%, respectively. Mice in CSP groups had 50% mortality, independent of the repeat-dosing protocol. We recommend redosing mice with either 50% of the initial ketamine dose or 25% of the initial ketamine-xylazine dose immediately upon return of the pedal withdrawal reflex to extend the surgical plane of anesthesia in mice, optimize the extension of the surgical plane, and minimize mortality.


Asunto(s)
Acepromazina/administración & dosificación , Anestésicos/administración & dosificación , Ketamina/administración & dosificación , Ratones Endogámicos C57BL , Xilazina/administración & dosificación , Animales , Relación Dosis-Respuesta a Droga , Inyecciones Intraperitoneales , Masculino , Ratones
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