Detection of Herpes Simplex Virus Type 1 in Human Ciliary Ganglia

Abstract

purpose. To determine whether herpes simplex virus type 1 (HSV-1) DNA is present in the ciliary ganglion (CG).

methods. Fifty CG and 47 trigeminal ganglia (TG) were resected from 63 formalin-fixed cadavers between 56 and 98 years of age that had been embalmed within 12 hours of death. The donors had no known active HSV infection at the time of death. DNA was extracted from all ganglia by proteinase-K digestion (TG) or digestion by a mild lysis buffer (CG). DNA was amplified by polymerase chain reaction for sequences from human chromosome 18, D18S1259 (positive control), and from the HSV-1 DNA polymerase gene, U_L30. The amplified DNA was separated by agarose gel electrophoresis, transferred to nylon membranes, and hybridized with the appropriate digoxigenin-labeled probe that was detected by alkaline phosphatase-conjugated monoclonal antibody.

results. The D18S1259 sequence was amplified from 47 TG and 30 CG samples. Of these samples, 32 (68.0%) of the 47 TG samples and 20 (66.6%) of the 30 CG samples were positive for the UL₃₀ HSV-1 sequence.

conclusions. Using amplification of HSV-1 DNA as a surrogate marker of latency, the finding that the frequency of HSV-1 in the CG was approximately the same as that of the TG suggests that the CG may be an additional site of HSV-1 latency in humans. Active infection in or reactivation of HSV-1 from non-TG sites may explain why this virus is able to infect sites, such as the retina, that have no direct connections to the trigeminal nerve.

Herpes simplex virus type 1 (HSV-1) has been implicated as the cause of several ocular diseases, including the acute retinal necrosis (ARN) syndrome, a relatively rare, but devastating, form of viral retinitis. ^{1 2} The ARN syndrome is a severe ocular inflammation characterized by the clinical triad of vitritis, occlusive vasculopathy, and progressive retinal necrosis. ³ Rhegmatogenous retinal detachment and blindness may also occur. ^{4 5} One or both eyes may be affected, although the time between involvement of the infected eye and the fellow eye may be weeks, months, or even years. ⁶ In contrast to other retinal necrotizing diseases, such as progressive outer retinal necrosis and cytomegalovirus (CMV) retinitis, ARN usually occurs in healthy, immunocompetent individuals. ⁷ One of the major questions related to the pathogenesis of this disease is how the virus gains access to the retina. 

Several animal models have been developed that are applicable for investigating the pathogenesis of the ARN syndrome in human patients. Acute necrotizing retinitis, a disease that parallels the ARN syndrome in several ways, can be induced experimentally in animals by uniocular anterior chamber (AC) inoculation of HSV-1. In the mouse, uniocular AC inoculation of HSV-1 results in acute, virally mediated retinitis only in the uninoculated contralateral eye 8 to 10 days post inoculation (PI). ^{8 9} Results from studies using rabbits inoculated with HSV-1 through the AC route indicate that HSV-1 gains access to the contralateral eye through the brain, ¹⁰ a finding that was confirmed by tracing studies in the mouse to determine the route of virus spread from the AC of the injected eye to the retina of the uninoculated eye. ¹¹ Sequentially, the path of virus spread in the mouse involves the following synaptically connected sites: AC of the inoculated eye (day 0), ciliary ganglion (CG) ipsilateral to the site of injection (day 2 PI), ipsilateral Edinger-Westphal nucleus (day 3 PI), ipsilateral suprachiasmatic nucleus (day 5 PI), contralateral optic nerve and retina (day 7 PI). ¹¹ Although the contralateral suprachiasmatic nucleus becomes virus positive (day 7 PI), virus does not spread from the contralateral suprachiasmatic nucleus to the ipsilateral optic nerve and retina. ¹¹  

Because the ARN syndrome in human patients and in animal models of HSV-1 ARN share several clinical manifestations, it has been suggested that the pattern of virus spread in humans may be similar to that observed in the mouse. ^{5 12} HSV-1 DNA has been recovered from several sensory and autonomic ganglia in humans, including the trigeminal (TG), spinal, vestibular, geniculate, and superior cervical ganglia. ^{13 14 15 16 17} However, information about the presence of HSV-1 in the CG, the ganglion that supplies postganglionic parasympathetic innervation to the anterior chamber, is unavailable. Because the CG, along with the TG and the trigeminal nerves, are the most likely to be involved in patients with herpes stromal keratitis who often have concomitant anterior uveitis, it was hypothesized that the CG may be a site of HSV-1 infection during acute infection and/or an additional site of latency after infection of sites synaptically connected to this ganglion. Reactivation of virus from this site followed by spread of virus into neuronal pathways synaptically connected to one or both optic nerves may be one mechanism by which ARN develops in humans. The purpose of these studies was to determine whether HSV-1 DNA is present in the CG of humans. 

Methods

Isolation of Trigeminal and Ciliary Ganglia

Trigeminal (TG) and ciliary ganglia (CG) were resected from formalin-fixed cadavers obtained from the Willed Body Program, The University of Texas Health Science Center at San Antonio (UTHSCSA), San Antonio, Texas. The cadavers used in this study ranged in age from 56 to 98 years. The ethnic and socioeconomic backgrounds of the cadavers were not available, although many donors were residents of the San Antonio metropolitan area, which has a large Hispanic population. The use of the human tissues described herein conformed to the tenets of the Declaration of Helsinki. None of the specimens was from a donor with a known active herpes virus infection, and the probable cause of death of most of the donors was cardiac or pulmonary disease (Table 1) . The sample pool of resected ganglia consisted of 49 TG and 52 CG collected from 63 cadavers. 

Because the ganglia for this study were collected from cadavers used in anatomic science courses at the UTHSCSA, limited dissection of the areas near the ganglia had been performed. As shown in Table 1 , generally only one half of a hemisected head was available from each cadaver. Although the dura surrounding the TG had been removed during study of the cranial nerves, in most of the specimens, the ciliary ganglion had not been exposed during dissection of the orbit. To minimize possible cross-contamination, each ganglion was removed using separate sterile instruments and gloves, and individual samples were placed in separate vials that were stored at −70°C. Repetitive washings with PBS were performed before DNA extraction to further reduce the chance of amplifying contaminating DNA sequences. Additional factors that helped to reduce the chance of ipsilateral or contralateral intra- or intercadaver contamination were the distance of the ganglia from each other (CG in orbit, TG in middle cranial fossa), structures between the CG and TG that could serve as barriers (orbital plate of the frontal bone, fat, muscle, and/or dura), and no prior dissection of the posterior orbit near the CG. 

TG were separated from the surrounding dura mater and resected by making cuts anterolateral to the opening to Meckel’s cave at the point where each of the three divisions (ophthalmic-V₁, maxillary-V₂, and mandibular-V₃) branch from the ganglion. The CG, which measures approximately 1 × 2 mm, lies approximately 10 mm anterior to the optic foramen. This ganglion is located between the optic nerve and lateral rectus muscle and appears as a small swelling connected to the nasociliary nerve. ^{18 19} Locating and identifying the CG was more difficult, owing to its small size and inconspicuous physical features with respect to the surrounding orbital tissue. Because its yellowish-brown appearance made it difficult to distinguish the CG from the surrounding adipose tissue, posterior illumination of the area near the CG was used to locate the ganglion, which appeared as a small, brown piece of tissue about the size of the point of a ballpoint pen in the center of a pad of yellow adipose tissue. ¹⁸ Before removal, the identity of the CG was confirmed by locating both the proximal and distal nerve connections. 

Preparation of TG and CG

The TG were washed with PBS, finely minced with sterile dissection blades, frozen in liquid nitrogen, placed in foil packets, and crushed with a mallet to ensure adequate surface area for homogenization of the tissue. After the TG were crushed, the tissue fragments were collected in a fresh, sterile vial for subsequent DNA extraction. Because of their small size, the CG samples were not crushed but instead were washed with PBS and suspended in embedding medium (OCT; Tissue-Tek; Sakura FineTek, Torrance, CA) in disposable vinyl specimen molds (Tissue-Tek Cryomold, Intermediate size; Sakura FineTek, Torrance, CA). After suspension, the samples were frozen at −70°C for at least 1 hour. To obtain fine pieces of CG samples, the frozen CG were sectioned using a cryostat, and the frozen sections were collected into new sterile vials. 

Extraction of DNA from TG and CG

Cell lysates of TG samples were made using 200 μL of a solution containing (final concentrations) 100 μg/mL proteinase K (Roche Molecular Biochemicals, Indianapolis, IN), 20 mM Tris-HCl (pH 7.4), 20 mM EDTA (pH 8.0), and 1.0% SDS. Samples were digested overnight in a 50°C water bath and then stored at −20°C. To purify TG DNA, the TG samples were extracted once with an equal volume of buffered saturated phenol (pH 7.49–7.79; GibcoBRL, Grand Island, NY)-chloroform-isoamyl alcohol (25:24:1) and then once with an equal volume of chloroform-isoamyl alcohol (24:1). After each extraction, the solutions were vortexed briefly and centrifuged at 3000g. 

Because of the paucity of tissue present in the CG samples and the propensity for DNA loss during phenol extraction, the CG samples were not subjected to phenol extraction and ethanol precipitation. Instead, PCR was performed on CG cell lysates made using a one-step lysis buffer containing (final concentrations): 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 2.5 mM MgCl₂, 0.1 mg/mL gelatin, 0.45% Nonidet (NP)-40, 0.45% Tween-20, and glass distilled water. Each CG sample was combined with 200 μL of the one-step lysis buffer, vortexed, and placed in boiling water for 10 minutes. Afterward, the CG samples were placed on ice for 5 minutes and subsequently stored at −20°C. The amount of DNA in the TG or CG samples was determined using a DNA minifluorometer (TKO 100; Hoefer Scientific Instruments, San Francisco, CA). The amount of DNA in the TG samples ranged between 71 and 133 ng. The amount of DNA in the CG samples ranged between 432 and 1000 ng. 

Polymerase Chain Reaction

TG and CG samples were subjected to PCR amplification for human chromosome 18 (to ensure the amplifiability of the DNA) and for HSV-1 sequences. A human genomic primer set (Table 2) was used to amplify a 150-bp sequence of human chromosome 18. Primers encoding a single-copy, noncoding region of chromosome 18, D18S1259 (sequences obtained from Robin Leach, Department of Cellular and Structural Biology) were constructed by the Center for Advanced DNA Technologies, UTHSCSA, San Antonio, Texas. The HSV-1 primer set (from the U_L30 encoding the HSV-1 DNA polymerase; Table 2 ) used in the amplification of viral DNA from samples and controls resulted in a 90-bp product. ²⁰ The HSV-1 primer set did not amplify human CMV, murine CMV, varicella zoster virus, or Epstein-Barr virus DNA sequences. The minimum level of detection of the HSV-1 primer set was 20 pg (6083 copies) of HSV-1 DNA (not shown). 

PCR reaction conditions for the chromosome 18 primer set were as follows: initial denaturation was at 95°C for 5 minutes followed by an annealing cycle at 55°C for 1 minute and a primer extension cycle at 72°C for 1 minute. Forty-four additional identical cycles were performed, except that the denaturation cycle was limited to 1 minute. PCR reaction conditions for the HSV-1 primer set were as follows: initial denaturation was at 94°C for 5 minutes, followed by a 75-second annealing cycle at 55°C and a primer extension period for 95 seconds at 72°C (with a 2-second subsequent cycle autoextension). Twenty-nine additional identical cycles were performed, except that the denaturation cycle was limited to 75 seconds. A negative control (1× TE; 10 mm Tris-HCl, 1 mm EDTA, pH 8.0) was included in every TG and CG PCR assay. In addition to the negative control, 100 pg/μL HSV-1 (KOS strain in 1× TE) was also used in every TG and CG PCR assay as a positive control. 

PCR products from CG samples were separated electrophoretically in 1.5% agarose gels and PCR products from TG samples were separated in 4% 3:1 agarose gels (NuSieve Plus; FMC Bioproducts, Rockland, ME) containing 0.75 μg/mL ethidium bromide (GibcoBRL, Gaithersburg, MD) in 0.5× Tris-borate EDTA buffer (TBE; GibcoBRL). Gels were photographed and blotted to nylon membranes (Hybond-N+; Amersham Pharmacia Biotech, Piscataway, NJ). The identity of PCR products was confirmed by hybridization to digoxigenin-labeled probes. Before hybridization, the filters containing the bound DNA were soaked in a prehybridization solution (125.0 mL 20× SSC [final concentration, 5×], 5.0 g blocking reagent [final concentration 1.0% wt/vol; Roche Molecular Biochemicals], 0.5 g N-lauryl sarcosine [final concentration 0.1% wt/vol], 1.0 mL 10% SDS [final concentration 0.02%], and 368.5 mL glass-distilled water [GDW] at 42°C) for 1 hour. Afterward, the membranes were soaked overnight in this same solution to which 75 μL (1 μg/μL) of the appropriate (HSV-1 or D18S1259) digoxigenin-labeled probe was added. Both probes bound DNA sequences internal to the PCR primers, and both probes were labeled with digoxigenin-11-dUTP with terminal deoxynucleotidyl transferase. The membranes were washed, and the probes were allowed to bind to anti-digoxigenin-alkaline-phosphatase-conjugated monoclonal antibodies. Visualization of the labeled-probes bound to the target sequences of DNA was achieved using 75 mg/mL nitroblue tetrazolium salt (NBT) and 50 mg/mL 5-bromo-4-chloro-3-indolyl phosphate toluidine salt (BCIP; Sigma-Aldrich Co., St. Louis, MO). 

Results

Amplification of Chromosome 18 Sequences in TG and CG

A photograph of a representative dissection of a left TG is shown in Figure 1A . Of 48 TG samples obtained by dissection, 47 were positive for the D18S1259 sequence after PCR and hybridization analyses. Representative results of the TG hybridization assays for the D18S1259 sequence are shown in Figure 2A . 

A photograph illustrating the small size and structures surrounding the left CG is shown in Figure 1B . Fifty-two CG samples were obtained by dissection but two could not be studied. Of the remaining 50 samples subjected to PCR and hybridization analyses for the D18S1259 sequence, 30 samples yielded positive results. Representative results of the CG hybridization assays for the D18S1259 sequence are shown in Figure 2B . 

Amplification of HSV-1 Sequences in TG and CG

Thirty-two (68.0%) of 47 TG samples and 20 (66.6%) of 30 CG samples were positive for the portion of the U_L30 gene sequence amplified by the HSV-1 primers. HSV-1 hybridization results from representative TG samples are shown in Figure 3A , and hybridization results from representative CG samples are shown in Figure 3B . 

A summary of the results for all specimens from which amplifiable DNA was obtained is presented in Table 1 . HSV-1 DNA was detected in the CG and TG from the same side in 8 (12.7%) of the 63 cadavers. Three other cadavers also had a positive TG and a positive CG, but these ganglia were not ipsilateral. As indicated in Table 1 , specimens from the remaining 52 cadavers had one positive ganglion (TG or CG), no positive ganglia, or ganglia from which DNA could not be amplified. Although it would have been ideal to collect all four ganglia from each cadaver, the condition of the specimens after dissection and/or the use of material containing these structures for other studies precluded this on most cadavers. 

Discussion

The results of these studies demonstrate that HSV-1 DNA can be amplified from the TG and CG of formalin-fixed, nonembedded material dissected from individuals who have donated their remains for use in studies of human anatomy. Although these results also support the idea that HSV-1 may be latent in the CG, there are several aspects of these studies that require consideration. In interpreting the results of these studies, it should be remembered that an obstacle to this type of study is created by the rigorous formalin-based fixation methods used to preserve cadaveric material that will be used in dissection-based human anatomy courses. Formalin is a fixative that creates cross-links between protein and DNA resulting in DNA damage during the fixation process. ²¹ Tokuda et al. ²² suggested that DNase may also contribute to the DNA damage that occurs in formalin-fixed tissue resulting in a lower yield of DNA that would be expected from comparable unfixed material. Difficulties in molecular examination of formalin-fixed tissue have been reported, ^{23 24 25 26} and these reports were used to guide the design of the extraction and amplification procedures described herein. 

The design of primers to amplify the target DNA sequences was another critical determinant of the results of this investigation. To establish a primer set appropriate for the amplification of a control sequence, formalin-fixed human dorsal root ganglia were subjected to DNA extraction and amplification with several different sets of human genomic PCR primers. Ultimately, the primer set that yielded the best results on amplification (D18S1259) was the one that produced an amplification product of approximately 150 bp (not shown). This result is in agreement with a study of the effects of formalin fixation on the ability to extract DNA from preserved neuropathologic material in which Kosel and Graeber ²⁶ reported that, to increase DNA yield from PCR amplification, target amplification sequences should be shorter than 300 bp. 

Although these results suggest that HSV-1 may be latent in CG, there are several caveats about this study that should be considered in interpretation of these results. First, information about seropositivity of the donors was not available; therefore, the percentage of HSV-1 DNA found in the samples cannot be compared with the premortem immune status of the donor. Second, it is possible that there was disease unaccounted for by the cause of death that may have had some effect on the ability of HSV-1 to infect and remain latent within these ganglia. For example, the donor could have had an undiagnosed HSV-1 infection at the time of his or her death. Third, in this study, it was assumed that the presence of HSV-1 DNA in these ganglia was a marker of a latent infection. The gold standard marker for HSV-1 latency, however, is detection of the latency-associated transcripts (LAT), mRNAs produced in latently infected neurons that play a role in the establishment and/or maintenance of the latent state. ^{27 28 29 30} In these studies, the presence of HSV-1 in the TG or CG was presumed to be a surrogate marker for latency, because these tissues could not be evaluated for the presence of LAT. Therefore, conclusions about the percentage of latently infected ganglia must be made with caution. However, in a recent report in which PCR was used to quantify the number of HSV-1 (and varicella-zoster virus) genomes in unfixed human trigeminal ganglia, the presence of viral DNA was equated with ganglionic latency. ³¹ Therefore, the presence of viral DNA is also likely to equate with latency in these studies. Finally, the percentage of ganglia positive for HSV-1 sequences may be underestimated in formalin-fixed cadaveric material because of the fixation process. However, it is likely that the fixation process would result in a similar amount of DNA cross-linking and/or DNA damage in all samples. 

Among studies performed in which unfixed TG samples collected at autopsy were examined for HSV-1 DNA, the percentage of positive samples ranged from 55% to 94%. ^{13 16 17 31 32} Because the results of TG positivity in this investigation using fixed material were within the range of HSV-1 positivity observed in other studies in which fresh TG material was used, these results suggest that the methods to extract and amplify DNA from formalin-fixed cadaveric tissues prepared for this study were appropriate and that the results are an accurate indication of the number of ganglia containing HSV-1 DNA. 

Before this investigation, HSV-1 DNA had not been reported in human CG samples. This study demonstrated that DNA could be extracted from formalin-fixed human CG and that the percentage of HSV-1 DNA-positive CG from this investigation was similar to the percentage of positive TG ganglia in this and other studies. ^{13 16 17 31 32} Possible explanations for the existence of HSV-1 DNA in the CG samples are (1) CG latency established after primary infection of AC structures innervated by oculomotor parasympathetic nerve fibers; (2) autoinoculation of the eye by fingers or other objects carrying HSV-1 from primary or recurrent oral HSV-1 lesions; (3) transfer of reactivated virus directly from the TG to the CG through the nasociliary nerve; and/or (4) transfer of HSV-1 from latent TG virus that reactivated, traveled to the nasociliary nerve endings, and subsequently infected nearby parasympathetic nerve fibers. This latter possibility is an indirect route by which HSV-1 latency could be established in the CG. However, regardless of the manner in which virus gains access to the CG, the percentage of positive CG samples in this study supports the idea that the CG may be another site of HSV-1 latency in addition to the TG. ³³  

In most cases, the ARN syndrome occurs in persons with no evidence of extraocular herpetic infection and who are generally considered healthy. ⁵ In spite of this, most cases of ARN syndrome appear to result from infections caused by reactivated herpes virus. ⁵ If the TG is the only repository for latent HSV-1 and assuming the virus has access to all three divisions of the trigeminal nerve, it would be reasonable to expect that ARN would be also accompanied by oral and facial lesions. The data from this investigation, however, provide a possible explanation for the absence of extraocular herpetic lesions in typical ARN cases resulting from reactivated HSV-1. Because the CG has no neural connections to the mouth or the face, reactivation of latent HSV-1 in a CG would probably produce symptoms primarily associated with the brain and/or eye, rather than the mouth or face. Although studies in the mouse have shown that HSV-1 can spread from structures in the anterior segment of the eye to the CG, it is not known whether HSV-1 in the anterior segment of human patients spreads to the CG as it does in the mouse. ¹¹  

In conclusion, even with the limitations discussed earlier, the results of these studies support the idea that HSV-1 is present in cell bodies other than those of the TG and that the CG may be an additional site of HSV-1 latency. However, it remains to be determined whether virus can reactivate from a non-TG site and whether it can reactivate if it infects neurons synaptically connected directly or indirectly to the eye and/or to other nonocular sites. 

 Supported by National Institutes of Health Grant EY06012.

 Submitted for publication December 19, 2001; revised February 25, 2002; accepted March 1, 2002.

 Commercial relationships policy: N.

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Table 1.

 

Donor Information and HSV-1 PCR Results

Donor	Age	Sex	TG			CG			Probable Cause of Death
						L	R	L		R
1	90	F	NR	–	NR	NR	Congestive heart failure
2	81	F	NR	–	NR	NA	Gastric lymphoma
3	78	M	NR	+	NR	+	Arteriosclerotic cardiovascular disease
4	90	F	NR	+	NR	–	Myocardial rupture
5	85	F	NR	+	NR	NR	Congestive heart failure
6	82	F	NR	+	NR	NR	Breast carcinoma
7	90	M	NR	+	NR	–	Sepsis
8	70	F	NR	+	NR	NA	Chronic lymphocytic leukemia
10	82	M	NR	+	NR	NR	Ventricular tachycardia
11	79	M	NR	+	NR	NR	Myocardial infarction
12	78	F	+	+	n/a	NR	Atherosclerosis; coronary artery disease
14	89	F	NR	+	NR	NR	Congestive heart failure
15	90	F	NR	+	NR	NR	Hypertension
16	85	F	NR	NR	NR	–	Cardiac arrest
17	92	M	NR	NR	–	NR	Cerebral arteriosclerosis
18	61	M	NR	NR	NR	–	Hepatocellular cancer
19	96	F	NR	+	NR	NR	Aspiration pneumonia
20	86	M	NR	NR	+	NR	Myocardial infarction
21	78	M	NR	+	NR	NA	Chronic obstructive pulmonary disease
22	83	M	+	NR	+	NR	Metastatic carcinoma
23	64	F	NR	NR	NR	+	Diabetes mellitus
24	78	F	NR	+	NR	NA	Chronic obstructive pulmonary disease
25	98	F	NR	+	+	NR	Malignant pleural effusion
26	85	F	NR	–	NR	NR	End-stage liver disease
27	83	F	+	NR	–	NR	Pneumonia
28	88	F	NR	+	NR	NA	Cerebrovascular accident
29	77	F	–	NR	+	NR	Aspiration pneumonia
30	55	F	NR	+	–	NA	Lung cancer
32	80	F	NR	NR	+	NR	Atherosclerotic cardiovascular disease
33	80	M	NR	NR	+	NA	Congestive heart failure
34	90	F	–	NR	NA	NA	Congestive heart failure
35	71	F	NR	–	+	NR	Septic shock
36	70	M	NR	NR	+	NR	Arteriosclerotic cardiovascular disease
37	68	F	NR	+	–	NR	Arteriosclerotic cardiovascular disease
38	73	F	NR	–	NR	NA	Metastatic breast carcinoma
39	94	F	NR	NR	+	NR	Aspiration pneumonia
40	73	M	NR	+	NR	+	Small-cell cancer
41	90	F	NR	–	NR	NR	Congestive heart failure
42	80	M	+	NR	+	NR	Metastatic prostate cancer
43	95	M	NR	NR	NR	–	Pneumonia
44	83	F	NR	+	+	NR	Asthma
45	76	F	NR	+	NR	+	Pulmonary embolus
46	74	M	–	NR	NA	NR	Atherosclerotic coronary artery disease
50	86	F	–	–	NR	NA	Hypertension
51	82	F	NR	–	NR	NR	Cardiac arrhythmia
52	74	F	NR	–	NR	NR	Subdural hemorrhage
53	79	F	+	–	+	NR	Arteriosclerosis
54	67	F	NR	+	NR	NR	Torsade de pointes
55	56	F	NR	–	NR	NR	Liver failure
56	74	M	NR	+	NR	NR	Diabetes
57	87	F	+	NR	+	NR	End-stage Alzheimer disease
58	88	F	+	NR	+	NR	Acute renal failure
59	86	F	NR	NR	NR	–	Anemia
60	71	F	NR	+	NR	NR	Metastatic squamous cell cancer
61	75	F	NR	NR	+	NR	Chronic obstructive pulmonary disease
62	86	F	NR	NR	+	NR	Anemia
63	?*	?	NR	+	NR	NR	Unknown

 Samples 9, 13, 31, 47, 48, and 49: no amplification for chromosome 18. NR, sample not retrieved, NA, sample could not be amplified.

*  Information not available.

Table 2.

 

Primers and Probes for Human Chromosome 18 and HSV-1

Specificity		Sequence
Human chromosome 18	Primer 1	5′-CTTAATGAAAACAATGCCAGAGC-3′
	Primer 2	5′-TGCAAAATGTGGAATAATCTGG-3′
	Probe	5′-GGTGGTAATCGAGGGTTAGC-3′
HSV-1	Primer 1	5′-CATCACCGACCCGGAGAGGGAC-3′
	Primer 2	5′-GGGCCAGGCGCTTGTTGGTGTA-3′
	Probe	5′-CTTTGTCCTCACCGCCGAACTGAGCAG-3′

Figure 1.

 

Photograph of a representative left TG dissection (A) and of a representative left CG dissection (B). Structures in (A): A, internal carotid artery; B, oculomotor nerve; C, trochlear nerve; D, ophthalmic nerve (V₁); E, maxillary nerve (V₂); F, mandibular nerve (V₃); G, trigeminal ganglion; H, trigeminal nerve; and J, middle meningeal artery. Structures in (B): (★), ciliary ganglion; A, lateral rectus muscle; B, optic nerve (CNII); C, levator palpebrae superioris muscle; D, superior rectus muscle; E, inferior rectus muscle; F, inferior division of the oculomotor nerve (CNIII); G, short ciliary nerves; H, ophthalmic artery; and J, nasociliary nerve.

Figure 2.

 

Chromosome 18 hybridization of electrophoretically separated PCR products illustrating positive and negative samples from the TG (A) and CG (B). (A) Lane 1, positive control; lane 2, negative control; lane 3, (subject [Table 1 ]/side of dissection) 56L; lanes 4 and 5, 45R; lanes 6 to 9, 53R; lanes 10 to 12, 6R; lanes 13 to 15, 15R; lanes 16 and 17, 56R; lane 18, positive control. (B) Lane 1, molecular weight marker; lane 2, positive control; lane 3, negative control; lane 4, 13R; lane 5, 2R; lane 6, 16R; lane 7, 12L; lane 8, 8R; lane 9, 46L; lane 10, 18R; lane 11, 29L; lane 12, 33R; lane 13, 31L; lane 14, 34R; lane 15, 38R; lane 16, 34L; lane 17, 28R; lane 18, 48R; lane 19, positive control; lane 20, molecular weight marker.

Figure 3.

 

HSV-1 hybridization of electrophoretically separated PCR products showing representative positive and negative samples from the TG (A) and CG (B). (A) Lane 1, positive control; lane 2, (subject [Table 1 ]/side of dissection) 6R; lane 3, 49R; lane 4, 37R; lane 5, 45R; lane 6, 60R; lane 7, 2R; lanes 8 to 10, negative controls; lane 11, positive control. (B) Lane 1, positive control; lane 2, 40 R; lane 3, negative control; lane 4, 17L; lane 5, 43R; lane 6, 4R; lane 7, 58L; lane 8, 30R; lane 9, 57L; lane 10, 31R; lane 11, 33L; lane 12, 27L; lane 13, 22L; lane 14, 44L; lane 15, 61L; lane 16, 53L; lane 17, 36 L; lane 18, positive control.

The authors thank Vick Williams, MD, PhD (Director, UTHSCSA Willed Body Program), Ben Castilleja (Director, UTHSCSA Anatomic Services Department), and the donors for their contributions to the study, and Linda Y. Johnson, PhD, William Morgan, PhD, and Charles J. Gauntt, PhD, of UTHSCSA for helpful suggestions and guidance.