Plasma Rich in Growth Factors Membrane as Coadjuvant Treatment in the Surgery of Ocular Surface Disorders

To evaluate the safety and efficacy of the surgical use of plasma rich in growth factors fibrin membrane (mPRGF) in different ocular surface pathologies.Fifteen patients with different corneal and conjunctival diseases were included in the study. Patients were grouped according to the use of mPRGF as graft (corneal and/or conjunctival) or dressing; they were also grouped according to the surgical subgroup of intervention (persistent corneal ulcer [PCU], keratoplasty, superficial keratectomy, corneal perforation, and pterygium). Best corrected visual acuity, intraocular pressure (IOP), inflammation control time (ICT), mPRGF AT (PRGF membrane absorption time), and the healing time of the epithelial defect (HTED) were evaluated throughout the clinical follow-up time. Safety assessment was also performed reporting all adverse events.mPRGF showed a total closure of the defect in 13 of 15 patients (86.7%) and a partial closure in 2 patients (13.3%). The mean follow-up time was 11.14.2 (4.8-22.8) months, the mean ICT was 2.5 +/- 1.1 (1.0-4.0) months, the mean mPRGF AT was 12.4 +/- 2.0 (10.0-16.0) days, and for the global HTED the mean was 2.9 +/- 1.2 (1-4.8) months. Results showed an improvement in BCVA in all patients, with an overall improvement of 2.9 in Vision Lines. The BCVA significantly improved (P.05) throughout the clinical follow-up time. No adverse events were reported after mPRGF use.The mPRGF is effective and safe as coadjuvant treatment in surgeries related with ocular surface disorders, being an alternative to the use of amniotic membrane. The mPRGF accelerates tissue regeneration after ocular surface surgery thus minimizing inflammation and fibrosis.This study received funding from the Ministry of Economy and Competitiveness of the Spanish Government, within the project denominated SURFEYE (reference RTC-2014-2375-1)

Expression Patterns of the <i>Drosophila</i> Neuropeptide CCHamide-2 and Its Receptor May Suggest Hormonal Signaling from the Gut to the Brain

<div><p>The insect neuropeptides CCHamide-1 and -2 are recently discovered peptides that probably occur in all arthropods. Here, we used immunocytochemistry, <i>in situ</i> hybridization, and quantitative PCR (qPCR), to localize the two peptides in the fruitfly <i>Drosophila melanogaster</i>. We found that CCHamide-1 and -2 were localized in endocrine cells of the midgut of larvae and adult flies. These endocrine cells had the appearance of sensory cells, projecting processes close to or into the gut lumen. In addition, CCHamide-2 was also localized in about forty neurons in the brain hemispheres and ventral nerve cord of larvae. Using qPCR we found high expression of the CCHamide-2 gene in the larval gut and very low expression of its receptor gene, while in the larval brain we found low expression of CCHamide-2 and very high expression of its receptor. These expression patterns suggest the following model: Endocrine CCHamide-2 cells in the gut sense the quality of food components in the gut lumen and transmit this information to the brain by releasing CCHamide-2 into the circulation; subsequently, after binding to its brain receptors, CCHamides-2 induces an altered feeding behavior in the animal and possibly other homeostatic adaptations.</p> </div

qPCR data for the expression of the <i>D. melanogaster</i> CCHamide-1 gene (CG14358), the CCHamide-1 receptor gene (CG14593), the CCHamide-2 gene (CG14375), and CCHamide-2 receptor gene (CG30106) in different organs or body parts of larval and adult flies.

<p>Third instar larval brain (a); third instar larval gut (b); third instar larval carcass (= rest of the body without brain and gut) (c); adult male brain (d); adult male gut (e); adult male carcass (= rest of the body without brain and gut) (f); adult female brain (g); adult female gut (h); adult female carcass (= rest of the body without brain and gut) (i). At least 25 organs were pooled for each qPCR measurement. These pools are the same in (A)-(D). The mRNA concentrations in each panel are given relative to column b (b=1). Other conditions are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076131#pone-0076131-g008" target="_blank">Figure 8</a>. A. Expression of the <i>D. melanogaster</i> CCHamide-1 gene. B. Expression of the <i>D. melanogaster</i> CCHamide-1 receptor gene. C. Expression of the CCHamide-2 gene. D. Expression of the CCHamide-2 receptor gene. Statistical analyses showed that there is a significant difference (P < 0.001) between the columns a and b; d and e; and g and h in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076131#pone-0076131-g009" target="_blank">Figure 9C and Figure 9D</a>.</p

Endocrine cells in the body wall of the midgut of third instar <i>D. melanogaster</i> stained for CCHamide-2.

<p>A. An overview, showing numerous endocrine cells in the anterior part of the anterior midgut (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076131#pone-0076131-g003" target="_blank">Figure 3</a> for a schematic drawing). Scale bar = 100 µm. B. A closer view of the endocrine cells, showing protrusions (arrow) directed toward the lumen of the gut. Scale bar = 20 µm. C. Close-up of a single endocrine cell. The basal part of the cell is in contact with the body circulation (the border of the gut epithelial cells and body cavity is stippled). Note that these cells are 25-40 µm in length. Scale bar = 20 µm.</p

Cartoon of our proposed mechanism for the hormonal action of CCHamide-2 in third instar larvae.

<p>This hypothetical model is based on the sensory nature of the CCHamide-2 immunoreactive endocrine cells in the gut (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076131#pone-0076131-g001" target="_blank">Figure 1</a>) and on our findings that there is abundant expression of the CCHamide-2 receptor gene in the brain (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076131#pone-0076131-g009" target="_blank">Figure 9D</a>, a), while this expression is virtually absent in the gut (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076131#pone-0076131-g009" target="_blank">Figure 9D, b</a>), combined with the high CCHamide-2 peptide gene expression in the gut (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076131#pone-0076131-g009" target="_blank">Figure 9C, b</a>). This model predicts that the quality of the food in the gut lumen is sensed by the CCHamide-2 immunoreactive endocrine cells in the gut wall. These cells release the CCHamide-2 peptides into the circulation, after which they reach the brain and bind to the CCHamide-2 receptors. This binding starts a cascade that leads to an altered (adapted) feeding behavior of the animal. In addition to this long-distance hormonal CCHamide-2 signaling, there is a local (synaptic or paracrine) CCHamide-2 signaling in the larval CNS, possibly also associated with feeding.</p

Schematic drawing of the localizations of the CCHamide-2 immunoreactive neurons and neuropil in the CNS of third instar larvae.

<p>The drawing shows the two hemispheres and the ventral nerve cord. The central neuropil of the larval CNS (stained with a synapsin mouse monoclonal antibody, cf. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076131#pone-0076131-g006" target="_blank">Figure 6 and Figure 7</a>) is outlined by green lines and shades. Neuronal perikarya and neuropil are drawn in red. Weakly immunoreactive perikarya are drawn as open red symbols. The perikarya indicated by 1 and 2 in the right hemisphere are located dorsally in each hemisphere; the perikarya indicated by 4, and 5, and 6 are located in the ventral parts of the hemispheres. The neuropils indicated by 3 as red dots in the anterior parts of the central neuropil are located partially ventrally and partially medially between the levels of the neurons 1 and 4. All perikarya in the ventral nerve cord are located in the ventral part of this nerve cord. They belong to the three fused thoracic ganglia.</p

A double-labeling experiment as in Figure 6, but now showing the posterior part of the ventral nerve cord.

<p>A. A dorsal view of the dorsal plane of the ventral nerve cord stained with the synapsin monoclonal antibody, showing the two columns of neuropil of the ventral nerve cord (red). B. The same focal plane as in A, but now showing staining with the CCHamide-2 antiserum (green). C. Merge of A and B. Dorsally located CCHamide-2 immunoreactive processes can be seen projecting in a longitudinal orientation over the whole nerve cord. Scale bar = 20 µm.</p

Neurons in the larval CNS stained with CCHamide-2 antisera.

<p>These preparations are somewhat compressed, because of mounting without the application of spacer rings. These spacer rings were present in the preparations shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076131#pone-0076131-g006" target="_blank">Figure 6 and Figure 7</a>. For a schematic representation of the neurons and neuropil in the two larval brain hemispheres and ventral nerve cord, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076131#pone-0076131-g005" target="_blank">Figure 5</a>. A. Ventral view of one hemisphere of the brain, showing 7-8 immunoreactive neurons located in the central region (long arrows) and processes (neuropil) projecting to other regions of the brain (short arrow). This neuropil corresponds to the neuropil indicated by number 3 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076131#pone-0076131-g005" target="_blank">Figure 5</a>. A piece of the ring gland (asterisk) is also visible, as well as a piece of the ventral nerve cord (VNC). Scale bar = 50 µm. B. Ventral view of another brain hemisphere with the ventral nerve cord. At least 12 nerve cells (short arrows) can be seen here symmetrically ordered along the midline of the ventral nerve cord (VNC). Scale bar = 50 µm.</p