Application of an organotypic ocular perfusion model to assess intravitreal drug distribution in human and animal eyes.

Intravitreal (ITV) drug delivery is a new cornerstone for retinal therapeutics. Yet, predicting the disposition of formulations in the human eye remains a major translational hurdle. A prominent, but poorly understood, issue in pre-clinical ITV toxicity studies is unintended particle movements to the anterior chamber (AC). These particles can accumulate in the AC to dangerously raise intraocular pressure. Yet, anatomical differences, and the inability to obtain equivalent human data, make investigating this issue extremely challenging. We have developed an organotypic perfusion strategy to re-establish intraocular fluid flow, while maintaining homeostatic pressure and pH. Here, we used this approach with suitably sized microbeads to profile anterior and posterior ITV particle movements in live versus perfused porcine eyes, and in human donor eyes. Small-molecule suspensions were then tested with the system after exhibiting differing behaviours in vivo. Aggregate particle size is supported as an important determinant of particle movements in the human eye, and we note these data are consistent with a poroelastic model of bidirectional vitreous transport. Together, this approach uses ocular fluid dynamics to permit, to our knowledge, the first direct comparisons between particle behaviours from human ITV injections and animal models, with potential to speed pre-clinical development of retinal therapeutics.

Transpupillary collagen photocrosslinking for targeted modulation of ocular biomechanics.

The central vision-threatening event in glaucoma is dysfunction and loss of retinal ganglion cells (RGCs), thought to be promoted by local tissue deformations. Here, we sought to reduce tissue deformation near the optic nerve head by selectively stiffening the peripapillary sclera, i.e. the scleral region immediately adjacent to the optic nerve head. Previous scleral stiffening studies to treat glaucoma or myopia have used either pan-scleral stiffening (not regionally selective) or regionally selective stiffening with limited access to the posterior globe. We present a method for selectively stiffening the peripapillary sclera using a transpupillary annular light beam to activate methylene blue administered by retrobulbar injection. Unlike prior approaches to photocrosslinking in the eye, this approach avoids the damaging effects of ultraviolet light by employing red light. This targeted photocrosslinking approach successfully stiffened the peripapillary sclera at 6 weeks post-treatment, as measured by whole globe inflation testing. Specifically, strain was reduced by 47% when comparing treated vs. untreated sclera within the same eye (n = 7, p=0.0064) and by 54% when comparing the peripapillary sclera of treated vs. untreated eyes (n = 7, p<0.0001). Post-treatment characterization of RGCs (optic nerve axon counts/density, and grading), retinal function (electroretinography), and retinal histology revealed that photocrosslinking was associated with some ocular toxicity. We conclude that a transpupillary photocrosslinking approach enables selective scleral stiffening targeted to the peripapillary region that may be useful in future treatments of glaucoma.

Author Correction: Improving Stem Cell Delivery to the Trabecular Meshwork Using Magnetic Nanoparticles.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Improving Stem Cell Delivery to the Trabecular Meshwork Using Magnetic Nanoparticles.

Glaucoma is a major cause of blindness and is frequently associated with elevated intraocular pressure. The trabecular meshwork (TM), the tissue that primarily regulates intraocular pressure, is known to have reduced cellularity in glaucoma. Thus, stem cells, if properly delivered to the TM, may offer a novel therapeutic option for intraocular pressure control in glaucoma patients. For this purpose, targeted delivery of stem cells to the TM is desired. Here, we used magnetic nanoparticles (Prussian blue nanocubes [PBNCs]) to label mesenchymal stem cells and to magnetically steer them to the TM following injection into the eye's anterior chamber. PBNC-labeled stem cells showed increased delivery to the TM vs. unlabeled cells after only 15-minute exposure to a magnetic field. Further, PBNC-labeled mesenchymal stem cells could be delivered to the entire circumference of the TM, which was not possible without magnetic steering. PBNCs did not affect mesenchymal stem cell viability or multipotency. We conclude that this labeling approach allows for targeted, relatively high-efficiency delivery of stem cells to the TM in clinically translatable time-scales, which are necessary steps towards regenerative medicine therapies for control of ocular hypertension in glaucoma patients.

Composition of external setae during regeneration and transformation of the bilaterally asymmetric claws of the snapping shrimp, Alpheus heterochelis.

The paired thoracic chelipeds or claws of adult snapping shrimp, Alpheus heterochelis, are bilaterally asymmetric, consisting of an enlarged and elaborate, sound-producing major (snapper) claw and a much smaller minor (pincer) claw. These paired claws vary in the composition of their external sensilla. Both possess long serrulate and simple short setae but the snapper also have plumose setae and long serrulate setae on the plunger. The pincers differ in having short serrulate setae and, in males alone, a prominent fringe of plumoserrate setae. During regeneration of each claw type, these setal structures are gradually added over three molts to reach the pristine condition. The long serrulate and simple short setae appear first, being seen in intermolt limb buds and commonly in both claws. Setae exclusive to each claw, i.e., plumoserrate and short serrulate in the pincer and plumose and long serrulate on the plunger in the snapper, appear sparsely in either the regenerated 1st or 2nd postmolt claw, they proliferate in the subsequent 2nd or 3rd postmolt claw. Transformation of the pincer claw to the snapper type begins in the 1st postmolt stage with the loss of pincer setae and addition of snapper setae and is completed by the 3rd postmolt stage. Since changes in composition of the external sensilla are restricted to postmolt stages, the underlying hypodermis is presumably being remodeled during proecdysis.

Claw Transformation and Regeneration in Adult Snapping Shrimp: Test of the Inhibition Hypothesis for Maintaining Bilateral Asymmetry.

In the paired asymmetric claws of adult snapping shrimp, Alpheus heterochelis, the minor, or pincer, claw may transform into a major, or snapper, claw if the existing snapper claw is damaged or lost, implying that an intact snapper claw normally inhibits the contralateral pincer claw from advancing to a snapper. We find that the pincer-to-snapper advancement in external form occurs almost immediately after the snapper is lost even as late as the premolt stage. The transforming claw in turn inhibits the newly regenerating pincer claw from becoming a snapper, but if the dactyl of the transforming claw is cut, then snapper-based inhibition is removed and the contralateral claw may regenerate as a snapper, resulting in shrimp with paired snapper claws. However, damaging an established snapper claw will not allow another snapper claw to regenerate at the pincer site, implying that less inhibition is required to restrict a newly regenerating claw to a pincer than to arrest an existing pincer claw. Inhibition may be manifested largely in terms of quantity of innervation. Hence the greater innervation of the snapper side over the pincer side would inhibit the pincer side, accounting for the regeneration of paired claws in their previous configuration following loss of both claws. Loss of the paired claws in two consecutive molts retards their development so that both claws often appear as pincers, but in succeeding molts one usually differentiates into a snapper and bilateral asymmetry is restored. In contrast, shrimp with paired snapper claws retain this configuration over several molts unless one or both of the claws are lost; in that case, regeneration restores bilateral asymmetry. Thus, bilateral asymmetry of the paired claws of adult shrimp is governed by a strong intrinsic lateralizing mechanism in which the snapper claw inhibits the pincer from advancing to another snapper.

Regenerate Limb Bud Sufficient for Claw Reversal in Adult Snapping Shrimps.

The paired, bilaterally asymmetric snapper and pincer claws in the adult snapping shrimp Alpheus heterochelis were simultaneously autotomized at the beginning of an intermolt, and the resulting growth of the limb buds was characterized into several stages. At the next molt the limb buds emerged as newly regenerated claws of the same morphotype as their predecessors. Next, the paired claws were autotomized sequentially, with the second autotomy timed to different stages of limb bud growth at the first autotomy site. When the snapper is autotomized and a limb bud varying from stages 1 to 5 is allowed to develop at this site before the pincer is removed, the paired claws regenerate in their previous configuration. Similarly, claw asymmetry is retained when the pincer claw is removed first and an early limb bud (stage 1-2) is allowed to form at this site before the snapper is autotomized. However, claw asymmetry is reversed if an advanced limb bud (stage 3-5) is allowed to form at the pincer site before the snapper claw is removed. Under these conditions a snapper regenerates at the pincer site and a pincer at the snapper site. Because the limb bud at this pincer site regenerates as a snapper rather than a pincer, claw transformation has occurred, with the stage 3-5 limb bud substituting for an intact pincer. Therefore, the minimal requirement for pincer-to-snapper transformation is a stage 3-5 limb bud. We postulate that the newly transforming snapper claw restricts regeneration at the contralateral old snapper site to a pincer, thereby ensuring that claw bilateral asymmetry is present, albeit reversed.

Morphology and Behavior of an Unusually Flexible Thoracic Limb in the Snapping Shrimp, Alpheus heterochelis.

The second thoracic limb in the snapping shrimp, Alpheus heterochelis, is much thinner, more elongated and flexible, and has a larger ganglion than its serial homologs. The greater length and flexibility is largely due to one of the limb segments--viz., the carpus--which consists of five separate segments, rather than the single segment typical of the other limbs. Externally, the multi-segmented carpus is relatively free of cuticular projections except for scattered simple setae. The adjoining segments--the merus and the propus--are also smooth except for clusters of long simple setae on the pollex and dactyl. Internally, each of the carpal segments has three muscles--a bender, stretcher and rotator--all restricted to the distal half of the segment. In keeping with the sensillum-free exterior of the multisegmented carpus, only about 1000 axon profiles originate in the carpus out of a total of 6000 counted at the base of the ganglion. This total number is roughly half that found in the first thoracic limbs. Conversely, the number of axon profiles in the longitudinal connectives to the second thoracic ganglion is about 25% greater than that to the first thoracic ganglion and may partly account for the size difference between these two ganglia. In terms of their behavior, the second thoracic limbs are almost constantly active, mostly probing the substrate, and occasionally grooming various body parts. Part of the probing behavior consists of food foraging and retrieval, especially from concealed and hard-to-reach locations. Because of their flexibility, these limbs are particularly adept at such movements.

Muscle and Nerve Terminal Fine Structure of a Primitive Crustacean, the Cephalocarid Hutchinsoniella macracantha.

Abdominal muscles of the cephalocarid Hutchinsoniella macracantha resemble the striated muscle fibers of other crustaceans, having regularly aligned sarcomeres that average 5 µm in length; thick, wavy Z-lines; and orbits of eight thin filaments surrounding a thick filament. However, unlike most crustacean muscle fibers, the cephalocarid muscle fibers are not subdivided into myofibrils by elaboration of the longitudinally oriented sarcoplasmic reticulum. Consequently, elements of the transverse tubule and sarcoplasmic reticulum in the form of triads occur scattered over the entire fiber. Motor innervation is by means of scattered nerve terminals, populated with round synaptic vesicles, indicative of excitatory axons. By lacking myofibrils, the cephalocarid and ostracod muscle represents a much simpler condition than the myofibril-rich muscles of the other crustacean classes and signifies a primitive condition in its resemblance to the onycophoran muscle.