Management of Retinitis Pigmentosa via Platelet Rich Plasma or Combination with Electromagnetic Stimulation: Retrospective Analysis of One-year Results

Purpose To investigate whether the natural progression rate of retinitis pigmentosa can be decreased with subtenon autologous platelet rich plasma application alone or combination with retinal electromagnetic stimulation. Material and methods The study includes retrospective analysis of 60 retinitis pigmentosa patients. Patients constitute 3 groups with similar demographic characteristics. The combined management group consists of 20 retinitis pigmentosa patients (40 eyes) who received combined retinal electromagnetic stimulation and subtenon platelet rich plasma as Group1; The subtenon platelet rich plasma-only group consisted of 20 retinitis pigmentosa patients (40 eyes) as Group2; The natural course (control) group consists of 20 retinitis pigmentosa patients (40 eyes) who did not receive any treatment were classified as Group3. Horizontal and vertical ellipsoid zone width, fundus perimetry deviation index and best corrected visual acuity changes were compared within and between groups after one year follow up period. Results Horizontal ellipsoid zone percentage changes were detected +1 % in Group1, -2.85% in Group2, -9.36% in Group3 (Δp 1>2>3). Vertical ellipsoid zone percentage changes were detected +0.34 % in Group1, -3.05% in Group2, -9.09% in Group3 (Δp 1>2>3). Fundus perimetry deviation index percentage changes were detected +0.05% in Group1, -2.68% in Group2 and -8.78% in Group3 (Δp 1>2>3). Conclusion Platelet-rich plasma is a good source of growth factors, but its half-life is 4-6 months. Subtenon autologous platelet rich plasma might more effectively slow down photoreceptor loss when repeated as booster injections and combined with retinal electromagnetic stimulation.

Retinitis pigmentosa (RP) is a progressive outer retinal degeneration resulting from mutation in any of the 260 genes found in the photoreceptor (PR) or retinal pigment epithelium (RPE) [1]. The progression rate and findings of the disease are heterogeneous according to genetic mutation and heredity type. The initial symptom of the disease is usually night blindness (nyctalopia) beginning in childhood or adolescence. Narrowing of the visual field and legal blindness develops as the disease progresses [2][3][4]. If low grade inflammation is added, then the disease is complicated by cataracts, an epiretinal membrane, and macular edema [5]. In the fundus examination, the appearance of midperipheral bone spicule pigmentation is usually sufficient to diagnosis [1].
Developments in spectral domain optical coherence tomography (SD-OCT) enable detailed imaging of the sensorial retina and the ellipsoid zone. Ellipsoid zone (EZ) is an OCT image of the inner and outer segments of photoreceptor cells. Loss of EZ is the gold standard in the diagnosis and follow-up of RP [6,7]. Visual field (VF) monitoring and electroretinography (ERG) are indirect signs of EZ loss and correlated with EZ width (EZW) [6]. Mutations in PR or RPE disrupt the synthesis of some vital peptides and growth factors for photoreceptors [1].
Autologous platelet-rich plasma (aPRP) is a good source of growth factors. Platelets have more than 30 growth factors and cytokines in α-granules. These peptides regulate the energy cycle at the cellular level. They also control local capillary blood flow, neurogenesis, and cellular metabolism [8,9]. Subtenon aPRP application in the management of RP patients has been shown to be clinically effective [10].
Molecules smaller than 75 kD can passively move from the sclera to the suprachoroidal space. Electrical or electromagnetic iontophoresis is required for molecules larger than 75kD such as BDNF and IGF to pass through the sclera into the subretinal space [15][16][17].
The clinical efficacy of rEMS alone or in combination with subtenon aPRP has also been shown [11].
The aim of this study is to investigate whether the natural progression rate of RP can be decreased with subtenon aPRP application alone or combination with rEMS.

Material And Methods
The study includes retrospective analysis of 60 RP patients who were followed up at Ankara University Faculty of Medicine between 2017 and 2019. Ethical committee approval was obtained from the Ankara University Faculty of Medicine Clinical Research Ethics Committee (19-1293-18). The study was carried out in accordance with the 2013 Helsinki Declaration.
The best corrected visual acuity (BCVA) was recorded as letters on the ETDRS chart (Topcon CC 100 XP, Japan). The ellipsoid zone width (EZW) shows healthy photoreceptors and was measured horizontally and vertically on cross-sectional structural SD-OCT (RTVue XR ''Avanti'', Optovue, Fremont, CA, USA). A manual segmentation program was used for the measurement of EZW. Fundus perimetry deviation index (FPDI) records were examined in the 24/2 visual field of computerized perimetry records (Compass, CenterVue, Padova, Italy). The FPDI offers data explaining how many of the 100 flashing points can be seen correctly by the patient and what percentage of the visual field can be seen.
The total amount of 60 RP patients constitute 3 groups with similar demographic characteristics: Group 1: The combined management group consists of 20 RP patients (40 eyes) who received combined rEMS and aPRP. The rEMS was applied with a custom-designed helmet for 30 minutes just before the subtenon aPRP injection. These combined applications were repeated 3 times a month with a 2-week interval (loading dose). Then, two additional booster doses were applied with 6-month intervals. The course of the disease was evaluated by comparing the BCVA, EZW, and FDPI parameters recorded before the first application and within 3 months after the last application.

Results
The mean age was 33.0 (22-51 years) in Group 1, 32.6 (20-56 years) in Group 2, 31.7 (20-57 years) in Group 3. The mean follow-up time between the first measurements and the last measurements in all three groups was 13 months (12-15 months). There were no statistical differences between the groups in terms of age and follow-up times (p = 0.81).
The mean horizontal ellipsoid zone width (m-HEZW): Group 1 was 3.46 mm before combined management and 3.50 mm after the procedures. During the mean 13-month follow-up, this positive change was 1.0% on average (p = 0.10). In Group 2, the m-HEZW was 3.32 mm at the first measurement and 3.26 mm after the PRP injections. During the mean 13-month follow-up, the change was found to be -2.9% on average (p = 0.01). In Group 3, the m-HEZW was 3.32 mm at the initial examination and 3.03 mm at the last examination. Over the 13-month follow-up, this negative change was found to be -9.4% on average (p = 0.01). Tables 1-4 When Groups 1, 2, and 3 were compared by the Sidak test according to the HEZW, VEZW, and FDPI changes, the combined application of rEMS and subtenon aPRP significantly increases the three assessment parameters. Table 4 Discussion There are currently over 260 different genetic mutations known to cause retinitis pigmentosa. Genetic inheritance can be autosomal dominant (AD), autosomal recessive (AR), X-linked, mitochondrial, mosaicism, or sporadic patterns [1]. Thus, the prognosis is usually quite heterogeneous. Acquired factors such as nutrition, smoking, anemia, pregnancy, as well as long-term exposure to ultraviolet and blue light also affect the course of the disease [2][3][4]. Autosomal dominant inheritance shows the slowest progression with an average annual loss of 5% photoreceptors [20,21]. X-linked inheritance shows the fastest progression with an average annual loss of 15% of photoreceptors [21,22].
Knowledge about which genetic mutation affects the progression is increasing due to widespread genetic testing. The annual progression rate of retinitis pigmentosa was reported to be 5% in RHO gene mutation that was inherited as AD, and 15% in RPGR gene mutation inherited as X-linked [20][21][22]. The photoreceptors have cilia tubule functions that provide the transport of opsin and rhodopsin and can be impaired by X-linked mutationsthey can be distinguished by the presence of widespread lipofuscin deposits in the fundus 9 examination. The ciliopathy gene mutations have three-fold faster progression than nonciliopathy mutations [23]. Retinitis pigmentosa progresses with an average of 10% annual photoreceptor loss when AD, AR, X-linked, and mitochondrial inheritance patterns are collectively evaluated [6,24,25]. In our study, the annual photoreceptor loss rate was found to be 9.3% on average in the RP group without interventional procedures (Group 3, and the removal of metabolic waste that occurs in RPE [26][27][28][29]. The growth factors, peptides, and fragments required for these functions are encoded by over 260 genes in RPE. Mutations in any of these genes leads to progressive vision loss and progressive degeneration of the sensorial unit [1]. In particular, mutations that affect the conversion of glucose to adenosine triphosphate (ATP) lead to a condition in photoreceptor cells called sleep mode or dormant phase [30,31]. Cells in this state have more solid plasma-they are live but metabolically inactive [32]. The photoreceptors in the dormant phase can be metabolically reactive if neurotrophins and GFs can be delivered the microenvironment of the sensorial unit [33]. Neurotrophins and GFs are key molecules in the cellular energy cycle [34]. Prolonged dormant phase or conditions impairing sensorial unit homeostasis eventually lead to apoptosis and cell loss [33]. RPE forms the outer blood-retinal barrier with its tight connections. Defects in the external blood retinal barrier due to apoptosis disrupt the immune-protected state in the retina and lead to lowdensity inflammation in the sensory unit. Neuro-inflammation accelerates the apoptosis process and sensorial unit loss [5].
Our previous clinical and prospective study showed that subtenon injection of aPRP significantly increased the visual functions [10,11]. Clinical and preclinical studies Trk receptors. Tyrosine kinase receptors are commonly found around the limbus, extraocular muscle insertions, and the optic nerve [19]. Molecules smaller than 75 kD can pass through the sclera via passive transport to the suprachroidal space [17]. BDNF and IGF are key growth factors in PRP and are larger than 75 kD [9].
Repetitive electromagnetic stimulation increases the affinity and synthesis of Trk growth factor receptors on neural tissues [11][12][13][14]. rEMS also provides electromagnetic iontophoresis effect by changing the electrical charges of the scleral pores and the peptides. Electrical or electromagnetic iontophoresis accelerates passing the large molecules such as BDNF and IGF through the sclera [15][16][17]. rEMS creates hyperpolarization-depolarization waves in neurons, which increases neuro-transmission and capillary blood flow [18]. In Group 1, rEMS was applied along with aPRP, and we found the change in mean EZW rate to be 0.7% at the end of one-year versus baseline. This result suggests that rEMS increases the effects of aPRP. The combined use of rEMS and aPRP has synergistic effects to prevent photoreceptor loss and reactivate the photoreceptor cells in sleep (dormant) mode. The electromagnetic field used here is far below the safety limits set by the World Health Organization [39].
In our study, ellipsoid zone widths and FDPI ratios in visual field showed similar changes.
This proves that the visual field is related to the number of photoreceptors. The visual field is a subjective test and can be influenced by many parameters such as refractive error, media opacity, illumination intensity, the patient's current attention, learning curve etc [40]. The visual field test gives indirect data about the number and functions of photoreceptors. EZW is an objective parameter in tracking the number of photoreceptors, it is not affected by subjective situations. We believe that EZW can be used for diagnosis and follow-up as a substitute for visual field and electroretinography in most cases. In our opinion, EZW should be the gold standard diagnostic-follow-up criterion for RP.
In contrast to the visual field, the central visual acuity is affected too late in RP. Apoptosis occurring in photoreceptors in the periphery leads to Müller cell hypertrophy and ectopic synaptogenesis in the central 19-degree area. Due to the paracrine effects of Müller cells, the cone cells are not affected by apoptosis for a long time. Consequently, BCVA can remain stable for a long time [41]. In our study, BCVA in all three groups did not change during an average of 13 months follow-up.
Local and systemic adverse events related to rEMS and/or aPRP were not detected during the one-year follow-up. Patients did not describe any uncomfortable condition except for temporary light sensitivity (which may last several days due to aPRP injection) and headache (which may last several hours due to rEMS application).
This retrospective clinical study has some limitations. The annual progression rate of retinitis pigmentosa varies depending on the type of genetic mutation. However, this issue was not analyzed here because the genetic mutation analysis of each patient could not be performed. Inflammatory findings were observed in some genetic mutation types of RP or in some stages of the disease. There were no measurements such as a laser flare meter regarding how aPRP or combined procedures affect the inflammatory response. The progression rate of each genetic type and the effects of interventional procedures on inflammation are additional research topics.

Conclusion
Retinitis pigmentosa is a neurodegenerative genetic disorder with progressive photoreceptor loss. In recent years, growth factor injections, stem cell applications, or gene therapy options have come into clinical use to slow or stop disease progression.
Platelet-rich plasma is a good source of growth factors, but its half-life is 4-6 months.
aPRP might more effectively slow down photoreceptor loss when repeated as booster injections and combined with retinal electromagnetic stimulation.  Tables   Due to technical limitations, Tables 1 -4 are only available for download from the Supplementary Files section.

Figures
21 Figure 1 Horizontal EZWs of the retinitis pigmentosa patient receiving aPRP+ rEMS. (Table   1 Patient no 1). a: Before treatment: 3.56 mm b: The 13th month of follow-up post-treatment: 3.83 mm.
23 Figure 3 Visual field FPDI changes of the retinitis pigmentosa patient receiving aPRP+ rEMS. (Table 1 Patient  Visual field FPDI changes of the retinitis pigmentosa patient receiving aPRP+ rEMS. (Table 1 Patient  Horizontal EZWs of the retinitis pigmentosa patient receiving only aPRP. (Table 2 Patient no. 1). a: Before treatment: 2.23 mm b: The 13th month of follow-up posttreatment: 2.51 mm.
27 Figure 7 Visual field FPDI changes of the retinitis pigmentosa patient receiving only aPRP.
28 Figure 8 Visual field FPDI changes of the retinitis pigmentosa patient receiving only aPRP.
(  Vertical EZWs of the retinitis pigmentosa patient, natural course. (Table 3 Patient no. 1) a: Before treatment 7.97 mm b: The 13th month of follow-up posttreatment: 7.09 mm.

Figure 10
Visual field FPDI changes of the retinitis pigmentosa patient, natural course. (